Mammalian DNA mismatch repair genes MLH1 and PMS1

ABSTRACT

We have discovered two human genes, hMLH1 and hPMS1, each of which apparently encodes for a protein involved in DNA mismatch repair. The hMLH1 gene encodes for a protein which is homologous to the bacterial DNA mismatch repair protein MutL, and is located on human chromosome 3p21.3-23. We believe that mutations in the hMLH1 gene cause hereditary non-polyposis colon cancer (HNPCC) in some individuals based upon the similarity of the hMLH1 gene product to the yeast DNA mismatch repair protein MLH1, the coincident location of the hMLH1 gene and the HNPCC locus on chromosome 3, and hMLH1 missense mutations in affected individuals from a chromosome 3-linked HNPCC family. The human hPMS1 gene is homologous to the yeast DNA mismatch repair gene PMS1, and is located on human chromosome 7q. We believe that the hPMS1 gene is a strong candidate for HNPCC testing because the yeast proteins MLH1 and PMS1 have been shown to be involved in the same DNA repair pathway and because hMLH1 and hMSH2 have both been implicated in HNPCC families. The most immediate use for hMLH1 and hPMS1 will be in screening tests on individuals who are members of families which exhibit high frequencies of early onset cancer. We have also isolated and sequenced mouse MLH1 and PMS1 genes. We have produced chimeric mice with a mutant form of the PMS1 gene that will enable us to derive mice that are heterozygous or homozygous for mutation in mPMS1. These mice will be useful for cancer research. We have also produced and isolated antibodies directed to hPMS1 which are useful in assays to detect the presence of protein in tumor samples.

BACKGROUND

This invention was made with government support under Agreement No. GM 32741 awarded by the National Institute of Health in the General Sciences Division. The government has certain rights in the invention.

This application is a continuation-in-part from U.S. patent application Ser. No. 08/168,877, titled: STRUCTURES FOR AND METHODS OF USING HUMAN DNA MISMATCH REPAIR GENES MLH1 AND MLH2 TO SCREEN FOR CANCER RISK, filed on Dec. 17, 1993, abandoned, the entirety of which is hereby incorporated by reference.

Since filing application Ser. No. 08/168,877, applicants have changed the names of the genes. The "MLH1" gene in the parent application is now named "PMS1". The "MLH2" gene in the parent application is now named "MLH1".

In recent years, with the development of powerful cloning and amplification techniques such as the polymerase chain reaction (PCR), in combination with the rapidly accumulating body of information concerning the structure and location of numerous human genes and markers, it has become not only possible, but practical and advisable to collect and analyze samples of DNA or RNA from individuals who are members of families which are identified as exhibiting a high frequency of certain genetically transmitted disorders. For example, screening procedures are routinely used to screen for genes involved in sickle cell anemia, cystic fibrosis, fragile X chromosome syndrome and multiple sclerosis. For some types of disorders, early diagnosis can greatly improve the person's long-term prognosis by, for example, adopting an aggressive diagnostic routine, or by making life style changes if appropriate to either prevent or prepare for an anticipated problem.

Once a particular human gene mutation is identified and linked to a disease, development of screening procedures to identify high-risk individuals can be relatively straight forward. For example, after the structure and abnormal phenotypic role of the mutant gene are understood, it is a relatively simple matter to design primers for use in PCR to obtain amplified quantities of the gene from individuals for testing. However, initial discovery of a mutant gene, i.e., its structure, location and linkage with a known inherited health problem, requires substantial experimental effort and creative research strategies.

One approach to discovering the role of a mutant gene in causing a disease begins with clinical studies on individuals who are in families which exhibit a high frequency of the disease. In these studies, the approximate location of the disease-causing locus is determined indirectly by searching for a chromosome marker which tends to segregate with the locus. A principal limitation of this approach is that, although the approximate genomic location of the gene can be determined, it does not generally allow actual isolation or sequencing of the gene.

For example, Lindblom et al.³ reported results of linkage analysis studies performed with SSLP (simple sequence length polymorphism) markers on individuals from a family known to exhibit a high incidence of hereditary non-polyposis colon cancer (HNPCC). Lindblom et al. found a "tight linkage" between a polymorphic marker on the short arm of human chromosome 3 (3p21-23) and a disease locus apparently responsible for increasing an individual's risk of developing colon cancer. Even though 3p21-23 is a fairly specific location relative to the entire genome, it represents a huge DNA region relative to the probable size of the mutant gene. Searching for the disease locus, i.e., gene mutation, within chromosome 3p21-23 is approximately analogous to searching for a person in San Francisco without knowing their address.

At best, such linkage studies have only limited utility for screening purposes because in order to predict one person's risk, genetic analysis must be performed with tightly linked genetic markers on a number of related individuals in the family. It is often impossible to obtain such information, particularly if affected family members are deceased. Without knowing the gene's structure, it is not possible to sample, amplify, sequence and determine directly whether an individual carries the mutant gene.

Another approach to discovering a disease-causing mutant gene begins with design and trial of PCR primers, based on known information about the disease, for example, theories for disease state mechanisms, related protein structures and function, possible analogous genes in humans or other species, etc. The objective is to isolate and sequence candidate normal genes which are believed to sometimes occur in mutant forms rendering an individual disease prone. This approach is highly dependent on how much is known about the disease at the molecular level, and on the investigator's ability to construct strategies and methods for finding candidate genes.

Association of a mutation in a candidate gene with a disease must ultimately be demonstrated by performing tests on members of a family which exhibits a high incidence of the disease. The most direct and definitive way to confirm such linkage in family studies is to use PCR primers which are designed to amplify portions of the candidate gene in samples collected from the family members. The amplified gene products are then sequenced and compared to the normal gene structure for the purpose of finding and characterizing mutations. A given mutation is ultimately implicated by showing that affected individuals have it while unaffected individuals do not, and that the mutation causes a change in protein function which is not simply a polymorphism.

Another way to show a high probability of linkage between a candidate gene mutation and disease is by determining the chromosome location of the gene, then comparing the gene's map location to known regions of disease-linked loci such as the one identified by Lindblom et al. Coincident map location of a candidate gene in the region of a previously identified disease-linked locus may strongly implicate an association between a mutation in the candidate gene and the disease.

There are other ways to show that mutations in the gene candidate may be linked to the disease. For example, artificially produced mutant forms of the gene can be cloned into animals. Incidence of the disease in animals carrying the mutant gene can then be compared to animals with the normal genotype. Significantly elevated incidence of disease in animals with the mutant genotype, relative to normal animals, may support the theory that mutations in the candidate gene are sometimes responsible for occurrence of the disease in the real world.

One type of disease which has recently received much attention because of the discovery of disease-linkable gene mutations is Hereditary Nonpolyposis Colon Cancer (HNPCC).¹,2 Members of HNPCC families also display increased susceptibility to other cancers including endometrial, gastric and breast. Recent publications have disclosed the identity and role of DNA mismatch repair gene hMSH2 in HNPCC.¹,2,12 Approximately 10% of colorectal cancers are believed to be HNPCC. A significant fraction of HNPCC cancers are believed to be due to mutations in the hMSH2 gene. hMSH2 is one of several genes whose normal function is to identify, and correct DNA mispairs following each round of chromosome replication. When genes of the "DNA Mismatch Repair" pathway are defective, mistakes invariably made during DNA replication remain uncorrected resulting in a greatly increased rate of spontaneous mutation and early onset cancer. It is estimated that as many as 1/200 individuals carry a mutation in either the MSH2 gene or other related genes which encode for another protein in the same DNA mismatch repair pathway.

Based on our knowledge of DNA mismatch repair mechanisms in bacteria and yeast including conservation of mismatch repair genes, we reasoned that, in addition to hMSH2, other human DNA mismatch repair homologs should exist. Further, mutations in such homologs affecting protein function, would be likely to cause genetic instability, possibly leading to an increased risk of certain forms of human cancer. Therefore, an important objective of our work has been to identify other candidate human genes which are useful for screening and identifying individuals who are at elevated risk of developing cancer.

SUMMARY OF THE INVENTION

We have isolated and sequenced two human genes, hPMS1 and hMLH1 each of which we believe encodes for a protein involved in DNA mismatch repair. Our studies strongly support an association between a mutation in hMLH1 and susceptibility to HNPCC. We believe mutations in hPMS1 may also result in an elevated risk of cancer. We believe our characterization and localization of hMLH1 and hPMS1 will be useful in the diagnosis, prevention and treatment of cancer. The most immediate use will be in screening tests on individuals who are members of families which exhibit an unusually high frequency of early onset cancer, for example, HNPCC.

Here we describe the isolation, sequence characterization and chromosomal map positions of hMLH1 and hPMS1. Each of these genes, based upon its similarity to bacterial and yeast genes of known function, is likely to have a role in repairing DNA replication errors that invariably occur during each cell division. The DNA correction process appears to be highly conserved from bacteria to humans, and is commonly referred to as DNA mismatch repair. Based upon studies in bacteria and yeast and the recent finding that mutations in a mismatch repair gene underlie certain forms of human cancer, inheritance of a mutation affecting protein function in either hMLH1 or hPMS1 is expected to raise mutation rates, and therefore predispose such individuals to multiple forms of cancer.

Recent evidence from our lab places the location of hMLH1 at human chromosome 3p21-23. This is a region of the human genome that, based upon family studies, harbors a locus that predisposes individuals to HNPCC. Additionally, we have found a mutation in a conserved region of the hMLH1 cDNA in HNPCC-affected individuals from a Swedish family. The mutation is not found in unaffected individuals from the same family, nor is it a simple polymorphism. Our discovery of a cancer-linked mutation, in hMLH1, combined with the gene's map position which is coincident with a previously identified HNPCC-linked locus, plus the likely role of the hMLH1 gene in mutation avoidance makes the hMLH1 gene a prime candidate for underlying one form of common inherited human cancer, and a prime candidate to screen and identify individuals who have an elevated risk of developing cancer.

We have also isolated and sequenced mouse MLH1 (mMLH1) and PMS1 (mPMS1) genes. We believe mMLH1 and mPMS1 will be useful for constructing animal models to study cancer. For example, such models may be used to identify additional oncogenes and to study environmental effects on mutagenesis.

We have produced polyclonal antibodies directed to a portion of the protein encoded by mPMS1 cDNA These antibodies also react with hPMS1 protein and are useful for detecting the presence of the protein encoded by a normal hPMS1 gene. We are also producing monoclonal antibodies directed to hMLH1.

In addition to diagnostic and therapeutic uses for the genes, our knowledge of hMLH1 and hPMS1 can be used to search for other genes of related function which play a role in certain forms of human cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow chart showing an overview of the sequence of experimental steps we used to isolate, characterize and use human and mouse PMS1 and MLH1 genes.

FIG. 2 is an alignment of protein sequences for MutL homologs showing two highly-conserved regions (underlined) which we used to create degenerate PCR oligonucleotides for isolating additional MutL homologs. In FIG. 2, the MutL protein is SEQ ID NO: 5. The HexB protein is SEQ ID NO: 6. The PMS1 protein is SEQ ID NO: 7.

FIG. 3 shows the entire cDNA nucleotide sequence for the human MLH1 gene, and the corresponding predicted amino acid sequence for the human MLH1 protein. The underlined DNA sequences are the regions of cDNA that correspond to the degenerate PCR primers that were originally used to amplify a portion of the MLH1 gene (nucleotides 118-135 and 343-359). In FIG. 3, the cDNA sequence for the human MLH1 gene is SEQ ID NO: 8. The predicted amino acid sequence for the human MLH1 protein is SEQ ID NO: 9.

FIG. 4 shows the nucleotide sequences of two genomic segments of the human MLH1 gene. The first segment shows the genomic sequence surrounding exon corresponding to nucleotides 139-228 of the cDNA (uppercase). The second segment shows the genomic sequence surrounding exon corresponding to nucleotides 229-327 of the cDNA (uppercase). In FIG. 4, the genomic sequence including the exon corresponding to nucleotides 139-228 of the cDNA is SEQ ID NO: 10. The genomic sequence including the exon corresponding to nucleotides 229-327 of the cDNA is SEQ ID NO: 11.

FIG. 5 is an alignment of the predicted amino acid sequences for human and yeast MLH1 proteins. Amino acid identities are indicated by boxes and gaps are indicated by dashes. In FIG. 5, the predicted amino acid sequence for human MLH1 protein is SEQ ID NO: 12. The predicted amino acid for yeast MLH1 protein is SEQ ID NO: 13.

FIG. 6 is a phylogenetic tree of MutL-related proteins.

FIG. 7 is a two-panel photograph. The first panel (A) is a metaphase spread showing hybridization of the hMLH1 gene of chromosome 3. The second panel (B) is a composite of chromosome 3 from multiple metaphase spreads aligned with a human chromosome 3 ideogram. The region of hybridization is indicated in the ideogram by a vertical bar.

FIG. 8 is a comparison of sequence chromatograms from affected and unaffected individuals showing identification of a C to T transition mutation that produces a non-conservative amino acid substitution at position 44 of the hMLH1 protein.

FIG. 9 is an amino acid sequence alignment of the highly-conserved region of the MLH family of proteins surrounding the site of the predicted amino acid substitution. Bold type indicates the position of the predicted serine to phenylalanine amino acid substitution in affected individuals. Also highlighted are the serine or alanine residues conserved at this position in MutL-like proteins. Bullets indicate positions of highest amino acid conservation. For the MLH1 protein, the dots indicate that the sequence has not been obtained. Sequences were aligned as described below in reference to the phylogenetic tree of FIG. 6. The segment of affected human MLH1 is SEQ ID NO: 14. The segment of normal human MLH1 protein is SEQ ID NO: 15. The mouse MLH1 protein is SEQ ID NO: 16. The segment of S. cerevisiae MLH1 protein is SEQ ID NO: 17. The segment of S. cerevisiae PMS1 protein is SEQ ID NO: 18. The segment of E. coli MutL protein is SEQ ID NO: 19. The segment of S. typhimutium MutL protein is SEQ ID NO: 20. The segment of S. pneumoniae HexB protein is SEQ ID NO: 21.

FIG. 10 shows the entire nucleotide sequence for hPMS1. In FIG. 10, the nucleotide sequence for hPMS1 is SEQ ID NO: 22.

FIG. 11 is an alignment of the predicted amino acid sequences for human and yeast PMS1 protein. Amino acid identities are indicated by boxes and gaps are indicated by dashes. The predicted amino acid sequence for hPMS1 protein is SEQ ID NO: 23. The predicted amino acid sequence for yeast PMS1 protein is SEQ ID NO: 24.

FIG. 12 is a photograph of a composite of chromosome 7 from multiple metaphase spreads showing hybridization of hPMS1.

FIG. 13 is a partial nucleotide sequence of mMLH1 cDNA. In FIG. 13, the partial nucleotide sequence of mMLH1 cDNA is SEQ ID NO: 25.

FIG. 14 is a comparison of the predicted amino acid sequence for mMLH1 and hMLH1 proteins. The predicted amino acid sequence for mMLH1 is SEQ ID NO: 26. The predicted amino acid sequence for hMLH1 is SEQ ID NO: 27.

FIG. 15 The cDNA nucleotide sequence for mPMS1is SEQ ID NO: 28.

FIG. 16 is a comparison of the predicted amino acid sequences for mPMS1 and hPMS1 proteins. The predicted amino acid sequence for mPMS1 is SEQ ID NO: 29. The predicted amino acid sequence for hPMS1 is SEQ ID NO: 30.

References

The following publications are referred to by number in the text of the application. Each of the publications is incorporated here by reference.

1. Fishel, R., et al. Cell 75, 1027-1038 (1993).

2. Leach, F., et al. Cell 75, 1215-1225 (1993).

3. Lindblom, A., Tannergard, P. I., Werelius, B. & Nordenskjold, M. Nature Genetics 5, 279-282 (1993).

4. Prolla, T. A., Christie, D. M. & Liskay, R. M. Molec. and Cell. Biol. 14,407-415 (1994).

5. Strand, M. Prolla, T. A., Liskay, R. M. & Petes, T. D. Nature 365, 274-276 (1993).

6. Aaltonen, L. A., et al. Science 260, 812-816 (1993).

7. Han, H. J., Yanagisawa, A., Kato, Y., Park, J. G. & Nakamura, Y. Cancer 53, 5087-5089 (1993).

8. Ionov, Y., Peinado, M. A. , Malkhosyan, S., Shibata, D. & Perucho, M. Nature 363, 558-561 (1993).

9. Risinger, J. I. et al. Cancer 53, 5100-5103 (1993).

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12. Parsons, R., et al. Cell 75, 1227-1236 (1993).

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15. Bishop, D. K., Anderson, J. & Kolodner, R. D. PNAS 86, 3713-3717 (1989).

16. Kramer, W., Kramer, B., Williamson, M. S. & Fogel, S. J. Bacteriol. 171, 5339-5346 (1989).

17. Williamson, M. S., Game, J. C. & Fogel, S., Genetics 110, 609-646 (1985).

18. Prudhomme, M., Martin, B., Mejean, V. & Claverys, J. J. Bacteriol. 171, 5332-5338 (1989).

19. Mankovich, J. A., McIntyre, C. A. & Walker, G. C. J. Bacteriol. 171, 5325-5331 (1989).

20. Lichter, P., et al. Science 247, 64-69 (1990).

21. Boyle, A., Feltquite, D. M., Dracopoli, N., Housman, D. & Ward, D.C. Genomics 12, 106-115 (1992).

22. Lyon, M. F. & Kirby, M. C., Mouse Genome 91, 40-80 (1993).

23. Reenan, R. A. & Kolodner, R. D. Genetics 132, 975-85 (1992).

24. Latif, F. et al. Cancer Research 52, 1451-1456 (1992).

25. Naylor, S. L., Johnson, B. E., Minna, J. D. & Sakaguchi, A. Y. Nature 329, 451-454 (1987).

26. Ali, I. U., Lidereau, R. & Callahan, R. Journal of the National Cancer Institute 81, 1815-1820 (1989).

27. Higgins, D., Bleasby, A. & Fuchs, R. Comput. Apple Biosci. 8, 189-191 (1992).

28. Fields, S. & Song, O. K. Nature 340, 245-246 (1989).

29. Lynch, H. T., et al. Gastroenterology 104, 1535-1549 (1993).

30. Elledge, S. J., Mulligan, J. T., Ramer, S. W., Spottswood, M. & Davis, R. W. Proc. Natl. Acad. Sci. U.S.A. 88, 1731-1735 (1991).

31. Frohman, M. Amplifications, a forum for PCR users 1, 11-15 (1990).

32. Powell, S. M., et al. New England Journal of Medicine 329, 1982-1987 (1993).

33. Wu, D. Y., Nozari, G. Schold, M., Conner, B. J. & Wallace, R. B. DNA 8, 135-142 (1989).

34. Mullis, K. E. B. & Faloona, F. A. Methods in Enzymology 155, 335-350 (1987).

35. Bishop, T. D., Thomas, H. Cancer Sur. 9, 585-604 (1990).

36. Capecchi, M. R. Scientific American 52-59 (March 1994).

37. Erlich, H. A. PCR Technology, Principles and Applications for DNA Amplification (1989).

DESCRIPTION OF THE INVENTION

We have discovered two mammalian genes which are apparently involved in DNA mismatch repair. One of the genes, PMS1, encodes a protein which is homologous to the yeast DNA mismatch repair protein PMS1. We have mapped the locations of PMS1 to human chromosome 7q and to mouse chromosome 5, band G. The other gene, MLH1 (MutL Homolog) encodes a protein which is homologous to the bacterial DNA mismatch repair protein MutL. We have mapped the locations of MLH1 to human chromosome 3p21.3-23 and to mouse chromosome 9, band E.

Recent studies ¹,2 have demonstrated involvement of a human DNA mismatch repair gene homolog, hMSH2, on chromosome 2p in HNPCC. Based upon linkage data, a second HNPCC locus has been assigned to chromosome 3p21-23.³ Examination of tumor DNA from the chromosome 3-linked kindreds revealed dinucleotide repeat instability similar to that observed for other INPCC families⁶ and several types of sporadic tumors..sup. 7-10 Because dinucleotide repeat instability is characteristic of a defect in DNA mismatch repair, ⁵, 11, 12 we reasoned that HNPCC linked to chromosome 3p21-23 could result from a mutation in a second DNA mismatch repair gene.

Repair of mismatched DNA in Escherchia coli requires a number of genes including mutS, mutL and mutH, defects in any one of which result in elevated spontaneous mutation rates.¹³ Genetic analysis in the yeast Saccharomyces cerevisiae has identified three DNA mismatch repair genes: a mutS homolog, MSH2,¹⁴ and two mutL homologs, PMS¹⁶ and MLH1.⁴ Each of these three genes play an indispensable role in DNA replication fidelity, including the stabilization of dinucleotide repeats.⁵

We believe that hMLH1 is the HNPCC gene previously linked to chromosome 3p based upon the similarity of the hMLH1 gene product to the yeast DNA mismatch repair protein, MLH1,⁴ the coincident location of the hMLH1 gene and the HNPCC locus on chromosome 3, and hMLH1 missense mutations which we found in affected individuals from a chromosome 3-linked HNPCC family.

Our knowledge of the human and mouse MLH1 and PMS1 gene structures has many important uses. The gene sequence information can be used to screen individuals for cancer risk. Knowledge of the gene structures makes it possible to easily design PCR primers which can be used to selectively amplify portions of hMLH1 and hPMS1 genes for subsequent comparison to the normal sequence and cancer risk analysis. This type of testing also makes it possible to search for and characterize hMLH1 and hPMS1 cancer-linked mutations for the purpose of eventually focusing the cancer screening effort on specific gene loci. Specific characterization of cancer-linked mutations in hMLH1 and hPMS1 makes possible the production of other valuable diagnostic tools such as allele specific probes which may be used in screening tests to determine the presence or absence of specific gene mutations.

Additionally, the gene sequence information can be used, for example, in a two hybrid system, to search for other genes of related function which are candidates for cancer involvement.

The MLH1 and PMS1 gene structures are useful for making proteins which are used to develop antibodies directed to specific portions or the complete MLH1 and PMS1 proteins. Such antibodies can then be used to isolate the corresponding protein and possibly related proteins for research and diagnostic purposes.

The mouse MLH1 and PMS1 gene sequences are useful for producing mice that have mutations in the respective gene. The mutant mice are useful for studying the gene's function, particularly its relationship to cancer.

Methods for Isolating and Characterizing Mammalian MLH1 and PMS1 Genes.

We have isolated and characterized four mammalian genes, i.e., human MLH1 (hMLH1), human PMS1 (hPMS1), mouse MLH1 (mPMS1) and mouse PMS1 (mPMS1). Due to the structural similarity between these genes, the methods we have employed to isolate and characterize them are generally the same. FIG. 1 shows in broad terms, the experimental approach which we used to isolate and characterize the four genes. The following discussion refers to the step-by-step procedure shown in FIG. 1.

Step 1 Design of degenerate oligonucleotide pools for PCR

Earlier reports indicated that portions of three MutL-like proteins, two from bacteria, MutL and HexB, and one from yeast, PMS1 are highly conserved.¹⁶,18,19 After inspection of the amino acid sequences of HexB, MutL and PMS1 proteins, as shown in FIG. 2, we designed pools of degenerate oligonucleotide pairs corresponding to two highly-conserved regions, KELVEN and GFRGEA, of the mutL-like proteins. The sequences of the degenerate oligonucleotides which we used to isolate the four genes are:

5'-CTTGATTCTAGAGC(T/C)TCNCCNC(T/G)(A/G)AANCC-3' and

5'-AGGTCGGAGCTCAA(A/G)GA(A/G)(T/C)TNGTNGANAA-3'. The sequences of the degenerate oligonucleotides shown above are SEQ ID NOs: 1 and 2, respectively.

The underlined sequences within the primers are XbaI and SacI restriction endonuclease sites respectively. They were introduced in order to facilitate the cloning of the PCR-amplified fragments. In the design of the oligonucleotides, we took into account the fact that a given amino acid can be coded for by more than one DNA triplet (codon). The degeneracy within these sequences are indicated by multiple nucleotides within parentheses or N, for the presence of any base at that position.

Step 2 Reverse transcription and PCR on poly A+ selected MRNA isolated from human cells

We isolated messenger (poly A+ enriched) RNA from cultured human cells, synthesized double-stranded cDNA from the mRNA, and performed PCR with the degenerate oligonucleotides.⁴ After trying a number of different PCR conditions, for example, adjusting the annealing temperature, we successfully amplified a DNA of the size predicted (˜210 bp) for a MutL-like protein.

Step 3 Cloning and sequencing of PCR-generated fragments; identification of two gene fragments representing human PMS1 and MLH1.

We isolated the PCR amplified material (˜210bp) from an agarose gel and cloned this material into a plasmid (pUC19). We determined the DNA sequence of several different clones. The amino acid sequence inferred from the DNA sequence of two clones showed strong similarity to other known MutL-like proteins.⁴,16,18,19 The predicted amino acid sequence for one of the clones was most similar to the yeast PMS1 protein. Therefore we named it hPMS1, for human PMS1. The second clone was found to most closely resemble yeast MLH1 protein and was named, hMLH1, for human MLH1.

Step 4 Isolation of complete human and mouse PMS1 and MLH1 cDNA clones using the PCR fragments as probes

We used the 210bp PCR-generated fragments of the hMLH1 and hPMS1 cDNAs, as probes to screen both human and mouse cDNA libraries (from Stratagene, or as described in reference 30). A number of cDNAs were isolated that corresponded to these two genes. Many of the cDNAs were truncated at the 5' end. Where necessary, PCR techniques ³¹ were used to obtain the 5'-end of the gene in addition to further screening of cDNA libraries. Complete composite cDNA sequences were used to predict the amino acid sequence of the human and mouse, MLH1 and PMS1 proteins.

Step 5 Isolation of human and mouse, PMS1 and MLH1 genomic clones

Information on genomic and cDNA structure of the human MLH1 and PMS1 genes are necessary in order to thoroughly screen for mutations in cancer prone families. We have used human cDNA sequences as probes to isolate the genomic sequences of human PMS1 and MLH1. We have isolated four cosmids and one P1 clone for hPMS1, that together are likely to contain most, if not all, of the cDNA (exon) sequence. For hMLH1 we have isolated four overlapping λ-phage clones containing 5'-MLH1 genomic sequences and a P1 clone. PCR analysis using pairs of oligonucleotides specific to the 5' and 3' ends of the hMLH1 cDNA, clearly indicates that the P1 clone contains the complete hMLH1 cDNA information. Similarly, genomic clones for mouse PMS1 and MLH1 genes have been isolated and partially characterized (described in Step 8).

Step 6 Chromosome positional mapping of the human and mouse, PMS1 and MLH1 genes by fluorescence in situ hybridization

We used genomic clones isolated from human and mouse PMS1 and MLH1 for chromosomal localization by fluorescence in situ hybridization (FISH).²⁰,21 We mapped the human MLH1 gene to chromosome 3p21.3-23, shown in FIG. 7 as discussed in more detail below. We mapped the mouse MLH1 gene to chromosome 9 band E, a region of synteny between mouse and human.22 In addition to FISH techniques, we used PCR with a pair of hMLH1-specific oligonucleotides to analyze DNA from a rodent/human somatic cell hybrid mapping panel (Coriell Institute for Medial Research, Camden, N.J.). Our PCR results with the panel clearly indicate that hMLH1 maps to chromosome 3. The position of hMLH1 3p21.3-23 is coincident to a region known to harbor a second locus for HNPCC based upon linkage data.

We mapped the hPMS1 gene, as shown in FIG. 12, to the long (q) arm of chromosome 7 (either 7q11 or 7q22) and the mouse PMS1 to chromosome 5 band G, two regions of synteny between the human and the mouse.²² We performed PCR using oligonucleotides specific to hPMS1 on DNA from a rodent/human cell panel. In agreement with the FISH data, the location of hPMS1 was confirmed to be on chromosome 7. These observations assure us that our human map position for hMLH1 to chromosome 7q is correct. The physical localization of hPMS1 is useful for the purpose of identifying families which may potentially have a cancer linked mutation in hPMS1.

Step 7 Using genomic and cDNA sequences to identify mutations in hPMS1 and hMLH1 genes from HNPCC Families

We are currently analyzing samples collected from individuals in HNPCC families for the purpose of identifying mutations in hPMS1 or hMLH1 genes. Our approach is to design PCR primers based on our knowledge of the gene structures, to obtain exon/intron segments which we can compare to the known normal sequences. We refer to this approach as an "exon-screening".

Using cDNA sequence information we designed hPMS1 and hMLH1 specific oligonucleotides to delineate exon/intron boundaries within genomic sequences. The hPMS1 and hMLH1 specific oligonucleotides were used to probe genomic clones for the presence of exons containing that sequence. Oligonucleotides that hybridized were used as primers for DNA sequencing from the genomic clones. Exon-intron junctions were identified by comparing genomic with cDNA sequences.

Amplification of specific exons from genomic DNA by PCR and sequencing of the products is one method to screen HNPCC families for mutations.¹,2 We have identified genomic clones containing hMLH1 cDNA information and have so far determined several exon-intron junctions. We have designed two pairs of oligonucleotides for PCR, that specifically amplify two exons (corresponding to nucleotides 139-228 and 229-327 in the hMLH1 cDNA, as shown in FIG. 4). We are continuing to use our knowledge of the complete cDNA sequence to determine the remaining exon-intron boundaries.

We have used the exon-screening approach to examine the MLH1 gene of individuals from HNPCC families showing linkage to chromosome 3.³ As will be discussed in more detail below, we identified a mutation in the MLH1 gene of one such family, consisting of a C to T substitution. We predict that the C to T mutation causes a serine to phenylalanine substitution in a highly-conserved region of the protein. We are continuing to identify HNPCC families from whom we can obtain samples in order to find additional mutations in hMLH1 and hPMS1 genes.

We are also using a second approach to identify mutations in hPMS1 and hMLH1. The approach is to design hPMS1 or hMLH1 specific oligonucleotide primers to produce first-strand cDNA by reverse transcription off RNA. PCR using gene-specific primers will allow us to amplify specific regions from these genes. DNA sequencing of the amplified fragments will allow us to detect mutations.

Step 8 Design targeting vectors to disrupt mouse PMS1 and MLH1 genes in ES cells; study mice deficient in mismatch repair.

We constructed a gene targeting vector based on our knowledge of the genomic mouse PMS1 DNA structure. We used the vector to disrupt the PMS1 gene in mouse embryonic stem cells. ³⁶ The cells were injected into mouse blastocysts which developed into mice that are chimeric (mixtures) for cells carrying the PMS1 mutation. The chimeric animals will be used to breed mice that are heterozygous and homozygous for the PMS1 mutation. These mice will be useful for studying the role of the PMS1 gene in the whole organism.

Human MLH1

The following discussion is a more detailed explanation of our experimental work relating to hMLH1. As mentioned above, to clone mammalian MLH genes, we used polymerase chain reaction (PCR) techniques like those used to identify the yeast MSH1, MSH2 and MLH1 genes and the human MSH2 gene.¹,2,4,14 As template in the PCR, we used double-stranded cDNA synthesized from poly (A+) enriched RNA prepared from cultured primary human fibroblasts. The degenerate oligonucleotides were targeted at the N-terminal amino acid sequences KELVEN and GFRGEA (see FIG. 3), two of the most conserved regions of the MutL family of proteins previously described for bacteria and yeast.¹⁶,18,19 Two PCR products of the predicted size were identified, cloned and shown to encode a predicted amino acid sequence with homology to MutL-like proteins. These two fragments generated by PCR were used to isolate human cDNA and genomic DNA clones.

The oligonucleotide primers which we used to amplify human MutL-related sequences were 5'-CTTGATTCTAGAGC(T/C)TCNCCNC(T/G)(A/G)AANCC-3' SEQ ID NO: 1 and 5'- AGGTCGGAGCTCAA(A/G)GA(A/G)(T/C)TNGTNGANAA-3' SEQ NO 2. PCR was carried out in 50 μL reactions containing cDNA template, 1.0 mM each primer, 5 IU of Taq polymerase (C) 50 mM KCl, 10 mM Tris buffer pH 7.5 and 1.5 mM MgCl. PCR was carried out for 35 cycles of 1 minute at 94° C., 1 minute at 43° C. and 1.5 minutes at 62° C. Fragments of the expected size, approximately 212 bp, were cloned into pUC19 and sequenced. The cloned MLH1 PCR products were labeled with a random primer labeling kit (RadPrime, Gibco BRL) and used to probe human cDNA and genomic cosmid libraries by standard procedures. DNA sequencing of double-stranded plasmid DNAs was performed as previously described.¹

The hMLH1 cDNA nucleotide sequence as shown in FIG. 3 encodes an open reading frame of 2268 bp. Also shown in FIG. 3 is the predicted protein sequence encoded for by the hMLH1 cDNA. The underlined DNA sequences are the regions of cDNA that correspond to the degenerate PCR primers that were originally used to amplify a portion of the MLH1 gene (nucleotides 118-135 and 343-359).

FIG. 4 shows two genomic segments of the hMLH1 gene. The first sequence shows the nucleotide structure surrounding exon corresponding to nucleotides 139-228 of the cDNA. The second sequence shows the genomic structure surrounding exon corresponding to nucleotides 229-327 of the cDNA.

As shown in FIG. 5, the hMLH1 protein is comprised of 756 amino acids and shares 41% identity with the protein product of the yeast DNA mismatch repair gene, MLH1.⁴ The regions of the hMLH1 protein most similar to yeast MLH1 correspond to amino acids 11 through 317, showing 55% identity, and the last 13 amino acids which are identical between the two proteins. FIG. 5 shows an alignment of the predicted human MLH1 and S. cerevisiae MLH1 protein sequences. Amino acid identities are indicated by boxes, and gaps are indicated by dashes. The pair wise protein sequence alignment was performed with DNAStar MegAlign using the clustal method.²⁷ Pair wise alignment parameters were a ktuple of 1, gap penalty of 3, window of 5 and diagonals of 5. Furthermore, as shown in FIG. 14, the predicted amino acid sequences of the human and mouse Mull proteins show at least 74% identity.

FIG. 6 shows a phylogenetic tree of MutL-related proteins. The phylogenetic tree was constructed using the predicted amino acid sequences of 7 MutL-related proteins: human MLH1; mouse MLH1; S. cerevisiae MLH1; S. cerevisiae PMS1; E. coli; MutL; S. typhimurium MutL and S. pneumoniae HexB. Required sequences were obtained from GenBank release 7.3. The phylogenetic tree was generated with the PILEUP program of the Genetics Computer Group software using a gap penalty of 3 and a length penalty of 0.1. The recorded DNA sequence has been submitted to GenBank.

Human MLH1 Link to Cancer

As a first step to determine whether hMLH1 was a candidate for the HNPCC locus on human chromosome 3p21-23,³ we mapped hMLH1 by fluorescence in situ hybridization (FISH).²⁰,21 We used two separate genomic fragments (data not shown) of the hMLH1 gene in FISH analysis. Examination of several metaphase chromosome spreads localized hMLH1 to chromosome 3p21.3-23.

Panel A of FIG. 7 shows hybridization of hMLH1 probes in a metaphase spread. Biotinylated hMLH1 genomic probes were hybridized to banded human metaphase chromosomes as previously described.20,21 Detection was performed with fluorescein isothiocyanate (FITC)-conjugated avidin (green signal); chromosomes, shown in blue, were counterstained with 4'6-diamino-2-phenylindole (DAPI). Images were obtained with a cooled CCD camera, enhanced, pseudocoloured and merged with the following programs: CCD Image Capture; NIH Image 1.4; Adobe Photoshop and Genejoin Maxpix respectively. Panel B of FIG. 7 shows a composite of chromosome 3 from multiple metaphase spreads aligned with the human chromosome 3 ideogram. Region of hybridization (distal portion of 3p21.3-23) is indicated in the ideogram by a vertical bar.

As independent confirmation of the location of hMLH1 on chromosome 3, we used both PCR with a pair of hMLH1-specific oligonucleotides and Southern blotting with a hMLH1-specific probe to analyze DNA from the NIGMS2 rodent/human cell panel (Coriell Inst. for Med. Res., Camden, N.J., USA). Results of both techniques indicated chromosome 3 linkage. We also mapped the mouse MLH1 gene by FISH to chromosome 9 band E. This is a position of synteny to human chromosome 3p.²² Therefore, the hMLH1 gene localizes to 3p21.3-23, within the genomic region implicated in chromosome 3-linked HNPCC families.³

Next, we analyzed blood samples from affected and unaffected individuals from two chromosome-3 candidate HNPCC families ³ for mutations. One family, Family 1, showed significant linkage (lod score=3.01 at recombination fraction of 0) between HNPCC and a marker on 3p. For the second family, Family 2, the reported lod score (1.02) was below the commonly accepted level of significance, and thus only suggested linkage to the same marker on 3p. Subsequent linkage analysis of Family 2 with the microsatellite marker D3S1298 on 3p21.3 gave a more significant lod score of 1.88 at a recombination fraction of 0 (unpublished data). Initially, we screened for mutations in two PCR-amplified exons of the hMLH1 gene by direct DNA sequencing (FIG. 4). We examined these two exons from three affected individuals of Family 1, and did not detect any differences from the expected sequence. In Family 2, we observed that four individuals affected with colon cancer are heterozygous for a C to T substitution in an exon encoding amino acids 4169, which corresponds to a highly-conserved region of the protein (FIG. 9). For one affected individual, we screened PCR-amplified cDNA for additional sequence differences. The combined sequence information obtained from the two exons and cDNA of this one affected individual represents 95% (i.e. all but the first 116 bp) of the open reading frame. We observed no nucleotide changes other than the C to T substitution. In addition, four individuals from Family 2, predicted to be carriers based upon linkage data, and as yet unaffected with colon cancer, were found to be heterozygous for the same C to T substitution. Two of these predicted carriers are below and two are above the mean age of onset (50 years) in this particular family. Two unaffected individuals examined from this same family, both predicted by linkage data to be non carriers, showed the expected normal sequence at this position. Linkage analysis that includes the C to T substitution in Family 2 gives a lod score of 2.23 at a recombination fraction 0. Using low stringency cancer diagnostic criteria, we calculated a lod score of 2.53. These data indicate the C to T substitution shows significant linkage to the HNPCC in Family 2.

FIG. 8 shows sequence chromatograms indicating a C to T transition mutation that produces a non-conservative amino acid substitution at position 44 of the hMLH1 protein. Sequence analysis of one unaffected (top panels, plus and minus strands) and one affected individual (lower panels, plus and minus strands) is presented. The position of the heterozygous nucleotide is indicated by an arrow. Analysis of the sequence chromatographs indicates that there is sufficient T signal in the C peak and enough A signal in the G peak for the affected individuals to be heterozygous at this site.

To determine whether this C to T substitution was a polymorphism, we sequenced this same exon amplified from the genomic DNA from 48 unrelated individuals and observed only the normal sequence. We have examined an additional 26 unrelated individuals using allele specific oligonucleotide (ASO) hybridization analysis.³³ The ASO sequences which we used are:

5'-ACTTGTGGATTTTGC-3' and

5'-ACTTGTGAATTTTGC-3'. The ASO sequences shown above are SEQ ID NOs: 3 and 4, respectively.

Based upon direct DNA sequencing and ASO analysis, none of these 74 unrelated individuals carry the C to T substitution. Therefore, the C to T substitution observed in Family 2 individuals is not likely to be a polymorphism. As mentioned above, we did not detect this same C to T substitution in affected individuals from a second chromosome 3-linked family, Family 1.³ We are continuing to study individuals of Family 1 for mutations in hMLH1.

Table 1 below summarizes our experimental analysis of blood samples from affected and unaffected individuals from Family 2 and unrelated individuals.

                  TABLE 1     ______________________________________                    Number of Individuals with     FAMILY 2       C to T Mutation/     Status         Number of Individuals Tested     ______________________________________     Affected       4/4     Predicted Carriers                    4/4     Predicted Non-carriers                    0/2     Unrelated Individuals                     0/74     ______________________________________

Based on several criteria, we suggest that the observed C to T substitution in the coding region of hMLH1 represents the mutation that is the basis for HNPCC in Family 2.³ First, DNA sequence and ASO analysis did not detect the C to T substitution in 74 unrelated individuals. Thus, the C to T substitution is not simply a polymorphism. Second, the observed C to T substitution is expected to produce a serine to phenylalanine change at position 44 (See FIG. 9). This amino acid substitution is a non-conservative change in a conserved region of the protein (FIGS. 3 and 9). Secondary structure predictions using Chou-Fasman parameters suggest a helix-turn-beta sheet structure with position 44 located in the turn. The observed Ser to Phe substitution, at position 44 lowers the prediction for this turn considerably, suggesting that the predicted amino acid substitution alters the conformation of the hMLH1 protein. Therefore, we propose that hMLH1 represents a second DNA mismatch repair gene that is involved in HNPCC. At present, we have no direct evidence that the hMLH1 gene is involved in the correction of DNA mispairs. In bacteria and yeast, a mutation affecting DNA mismatch repair causes comparable increases in the rate of spontaneous mutation including additions and deletions within dinucleotide repeats.⁴,5,11,13,14,15,16 In humans, mutation of hMSH2 is the basis of chromosome-2 HNPCC,¹,2 tumors of which show microsaterlite instability and an apparent defect in mismatch repair.¹² Chromosome 3-linked HNPCC is also associated with instability of dinucleotide repeats.³ Combined with these observations, the high degree of conservation between the human MLH1 protein and the yeast DNA mismatch repair protein MLH1 suggests that hMLH1 is likely to function in DNA mismatch repair. During isolation of the hMLH1 gene, we identified the hPMS1 gene. This observation suggests that mammalian DNA mismatch repair, like that in yeast,⁴ may require at least two MutL-like proteins.

It should be noted that it appears that different HNPCC families show different mutations in the MLH1 gene. As explained above, affected individuals in Family 1 showed "tight linkage" between HNPCC and a locus in the region of 3p21-23. However, affected individuals in Family 1 do not have the C to T mutation found in Family 2. It appears that the affected individuals in Family 1 have a different mutation in their MLH1 gene. Unlike, for example, sickle cell anemia, in which essentially all known affected individuals have the same mutation genotype, it is likely that multiple hMLH1 mutations will be eventually linked to cancer. Therefore, knowledge of the entire cDNA sequence for hMLH1 (and probably hPMS1), as well as genomic sequences particularly those that surround exons, will be useful and important for characterizing mutations in families identified as exhibiting a high frequency of cancer.

In summary, we have described a second DNA mismatch repair gene homolog, hMLH1, which is likely to be the hereditary nonpolyposis colon cancer gene previously localized by linkage analysis to chromosome 3p21-23.³ Like other HNPCC families,²⁹,35 chromosome 3p-families show apparent predisposition to several types of cancer.³ The availability of the hMLH1 and hMSH2 gene sequences will facilitate the screening of HNPCC families for mutations in either gene. In addition, although loss of heterozygosity (LOH) of linked markers is not a feature of either the 2p or 3p forms of HNPCC,³,6 LOH involving the 3p21.3-23 region has been observed in several human cancers.24-26 This raises the possibility that hMLH1 mutation may play some role in these tumors. Finally, it seems likely that defects in additional DNA mismatch repair genes will prove to be the basis for the cancer in HNPCC families showing neither chromosome 2p or 3p involvement.³

Human PMS1

Human PMS1 was isolated using the procedures discussed with reference to FIG. 1. FIG. 10 shows the entire hPMS1 cDNA nucleotide sequence. FIG. 11 shows an alignment of the predicted human and yeast PMS1 protein sequences. FIG. 12 is a metaphase spread showing the results of FISH analysis to determine the map location of human PMS1. The results shown in FIG. 12 indicate two possible loci for human PMS1, namely 7q 11 and 7q 22.

Utility of the hMLH1 and hPMS1 Genes

Our recent map position and mutation data strongly suggest that the hMLH1 gene is the HNPCC locus that, based upon linkage studies, maps to chromosome 3p.³ The hPMS1 on 7q is, a priori, also a strong candidate to represent HNPCC that maps to neither 2p or 3p.1-3,6 At present, our collaborators in Sweden are checking such "unlinked" HNPCC families for linkage to 7q. Clearly, our data indicate that the hMLH1 gene has significant utility in the screening and diagnosis of hereditary human cancer. Further studies are required to fully determine the utility of the hPMS1 gene in human cancer risk screening. We would like to point out, however, that several observations make the hPMS1 gene a strong DNA mismatch repair gene candidate and hence a possible player in human cancer. These observations include: 1) the involvement of mismatch repair gene homologs, hMSH2 and hMLH1, in HNPCC;¹,2 2) the close similarity of the human and yeast PMS1 proteins 3) the role of the yeast PMS1 protein in DNA mismatch repair;⁴ and 4) our published genetic and unpublished biochemical data strongly suggesting that the yeast PMS1 and MLH1 proteins act as a heteromeric complex during DNA mismatch repair.

Use of MLH genes in the detection of individuals at risk for cancer

It has been estimated that approximately 1,000,000 individuals in the United States carry (are heterozygous for) an HNPCC mutant gene.²⁹ Furthermore, estimates suggest that 50-60% of HNPCC families segregate mutations in the MSH2 gene that resides on chromosome 2p.¹,2 Another significant fraction would appear to be associated with the HNPCC gene that maps to chromosome 3p21-22, presumably due to hMLH1 mutations such as the C to T transition discussed above. Identification of families that segregate mutant alleles of either the hMSH2 or HMLH1 gene, and the determination of which individuals in these families actually have the mutation will be of great utility in the early intervention into the disease. Such early intervention will likely include early detection through screening and aggressive follow-up treatment of affected individuals. In addition, determination of the genetic basis for both familial and sporadic tumors could direct the method of therapy in the primary tumor, or in recurrences.

Detection of HNPCC families and their mutation(s)

Initially, HNPCC candidate families will be diagnosed partly through the study of family histories, most likely at the local level, e.g., by hospital oncologists. One criterion for HNPCC is the observation of microsatellite instability in these individual's tumors.³,6 The presenting patient would be tested for mutations in hMSH2, hMLH1 and other genes involved in DNA mismatch repair as they are identified. This is most easily done by sampling blood from the individual. Also highly useful would be freshly frozen tumor tissue. Of importance to note for the screening procedure, the affected individuals are heterozygous for the offending mutation in their normal tissues.

The available tissues, e.g. blood and tumor, are worked up for PCR-based mutation analysis using one or both of the following procedures:

1) Linkage analysis with a microsatellite marker tightly linked to the hMLH1 gene.

One approach to identify cancer prone families with a hMLH1 mutation is to perform linkage analysis with a highly polymorphic marker located within or tightly linked to hMLH1. Microsatellites are highly polymorphic and therefore are very useful as markers in linkage analysis. Because we possess the hMLH1 gene on a single large genomic fragment in a P1 phage clone (˜100 kbp), it is very likely that one or more microsatellites, e.g. tracts of dinucleotide repeats, exist within, or very close to, the hMLH1 gene. Once such markers have been identified, PCR primers will be designed to amplify the stretches of DNA containing the microsatellites. DNA of affected and unaffected individuals from a family with a high frequency of cancer will be screened to determine the segregation of the MLH1 markers and the presence of cancer. The resulting data can be used to calculate a lod score and hence determine the likelihood of linkage between hMLH1 and the occurrence of cancer. Once linkage is established in a given family, the same polymorphic marker can be used to test other members of the kindred for the likelihood of their carrying the hMLH1 mutation.

2) Sequencing of reverse transcribed cDNA.

a) RNA from affected individuals, unaffecteds and unrelated individuals is reverse transcribed (R'd), followed by PCR to amplify the cDNA in 4-5 overlapping portions.34,37 It should be noted that for the purposes of PCR, many different oligonucleotide primer pair sequences may potentially be used to amplify relevant portions of an individual's hMLH1 or hPMS1 gene for genetic screening purposes. With the knowledge of the cDNA structures for the genes, it is a straight-forward exercise to construct primer pairs which are likely to be effective for specifically amplifying selected portions of the gene. While primer sequences are typically between 20 to 30 bases long, it may be possible to use shorter primers, potentially as small as approximately 13 bases, to amplify specifically selected gene segments. The principal limitation on how small a primer sequence may be used is that it must be long enough to bond specifically to the targeted gene segment.

These PCR products, in total representing the entire cDNA, are then sequenced and their sequences compared. In most cases a mutation will be observed in the affected individual. Ideally, the nature of mutation will indicate that it is likely to inactivate the gene product. Otherwise, the possibility that the alteration is not simply a polymorphism must be determined.

b) Certain mutations, e.g. those affecting splicing or resulting in translation stop codons, can destabilize the messenger RNA produced from the mutant gene and hence comprise the normal RT-based mutation detection method. One recently reported technique can circumvent this problem by testing whether the mutant cDNA can direct the synthesis of normal length protein in a coupled in vitro transcription/translation system.³²

3) Direct sequencing of the genomic DNA.

A second route to detect mutations relies on examining the exons and the exon/intron boundaries by PCR cycle sequencing directly off a DNA template.¹,2 This method requires the use of oligonucleotide pairs that amplify individual exons for direct PCR cycle sequencing. The method depends upon genomic DNA sequence information at each intron/exon boundary (50 bp, or greater, for each boundary). The advantage of the technique is two fold. First, because DNA is more stable than RNA, the condition of the material used for PCR is not as important as it is for RNA-based protocols. Second, most any mutation within the actual transcribed region of the gene, including those in an intron affecting splicing, should be detectable.

Therefore, for each candidate gene, mutation detection may require knowledge of both the entire cDNA structure, and all intron/exon boundaries of the genomic structure. With such information, the type of causal mutation in a particular family can be determined. In turn, a more specific and efficient mutation detection scheme can be adapted to that family. In many cases such a "family by family" delineation will most likely be beneficial for HNPCC. The disease is not only genetically heterogeneous in the sense that more than one gene is involved, but also because for a particular gene, multiple types of mutations are involved.² Any given family is highly likely to segregate one particular mutation. However, as the nature of the mutation in multiple families is determined, the spectrum of the most prevalent mutations in the population can be determined. In general, determination of the most frequent mutations will direct and streamline mutation detection.

Because HNPCC is so prevalent in the human population, carrier detection at birth could become part of standardized neonatal testing. Families at risk would be identified and all members not previously tested would be tested. Eventually, all affected kindreds could be determined.

Mode of mutation screening and testing

DNA-based Testing

Some of the initial testing, including detecting likely HNPCC families by standard diagnosis and family history study, will likely be done in local and smaller DNA diagnosis laboratories. However, large scale testing of multiple family members, and certainly population wide testing, will ultimately require large efficient centralized commercial facilities. The exact method of large scale screening at such facilities is not clear but is likely to involve at least some of the methods and techniques described above.

It seems likely that tests will be developed based on the determination of the most common mutations for the two major genes behind HNPCC, the hMSH2 gene on chromosome 2p and the MLH1 gene on chromosome 3p. A variety of tests are likely to be developed. For example, one possibility is a set of tests employing oligonucleotide hybridizations that distinguish the normal vs. mutant alleles.³³ As already noted, our knowledge of the nucleotide structures for hMLH1, hPMS1 and hMSH2 genes makes possible the design of numerous oligonucleotide primer pairs which may be used to amplify specific portions of an individual's mismatch repair gene for genetic screening and cancer risk analysis. Our knowledge of the genes' structures also makes possible the design of labeled probes which can be quickly used to determine the presence or absence of all or a portion of one of the DNA mismatch repair genes. For example, allele-specific oligomer probes (ASO) may be designed to distinguish between alleles. ASOs are short DNA segments that are identical in sequence except for a single base difference that reflects the difference between normal: and mutant alleles. Under the appropriate DNA hybridization conditions, these probes can recognize a single base difference between two otherwise identical DNA sequences. Probes can be labeled radioactively or with a variety of non-radioactive reporter molecules, for example, fluorescent or chemiluminescent moieties. Labeled probes are then used to analyze the PCR sample for the presence of the disease-causing allele. The presence or absence of several different disease-causing genes can readily be determined in a single sample. The length of the probe must be long enough to avoid non-specific binding to nucleotide sequences other than the target. All tests will depend ultimately on accurate and complete structural information on the MLH1 gene on chromosome 3p and the MutS homolog, hMSH2, on chromosome 2p.

Protein Detection-based Screening

Although it is likely that most screening tests will be initially DNA-based, tests based on the status of the protein product, per se, will also have utility. These protein-examining tests will most likely utilize antibody reagents specific to either the hMLH11, hPMS1 and hMSH2 proteins or other related "cancer" gene products as they are identified.

For example, a frozen tumor specimen can be cross-sectioned and prepared for antibody staining using indirect fluorescence techniques. Certain gene mutations are expected to alter or destabilize the protein structure sufficiently such as to give an altered or reduced signal after antibody staining. It is likely that such tests would be performed most often in cases where gene involvement in a family's cancer has not yet been established. Therefore, the development of diagnostic monoclonal antibodies against the human MLH1 and PMS1 proteins is a high priority in our laboratory. We are overexpressing each of these human proteins in bacteria. We will purify the proteins, inject them into mice and derive protein specific monoclonal antibodies which can be used for diagnostic and research purposes.

Use of hPMS1 and hMLH1 cDNAs for Isolating Additional Genes of Related Function

Proteins that interact physically with either hMLH1 and/or hPMS1, are likely to be involved in DNA mismatch repair. By analogy to hMLH1 and hMSH2, mutations in the genes which encode for such proteins would be strong candidates for potential cancer linkage. A powerful molecular genetic approach using yeast, referred to as a "two-hybrid system", allows the relatively rapid detection and isolation of genes encoding proteins that interact with a gene product of interest, e.g. hMLH1.²⁸

The two-hybrid system involves two plasmid vectors each intended to encode a fusion protein. Each of the two vectors contains a portion, or domain, of a transcription activator. The yeast cell used in the detection scheme contains a "reporter" gene. The activator alone cannot activate transcription. However, if the two domains are brought into close proximity then transcription may occur. The cDNA for the protein of interest, e.g., hMLH1 is inserted within a reading frame in one of the vectors. This is termed the "bait". A library of human cDNAs, inserted into a second plasmid vector so as to make fusions with the other domain of the transcriptional activator, is introduced into the yeast cells harboring the "bait" vector. If a particular yeast cell receives a library member that contains a human cDNA encoding a protein that interacts with hMLH1 protein, this interaction will bring the two domains of the transcriptional activator into close proximity, activate transcription of the reporter gene and the yeast cell will turn blue. Next, the insert is sequenced to determine whether it is related to any sequence in the data base. The same procedure can be used to identify yeast proteins in DNA mismatch repair or a related process. Performing the yeast and human "hunts" in parallel has certain advantages. The function of novel yeast homologs can be quickly determined in yeast by gene disruption and subsequent examination of the genetic consequences of being defective in the new found gene. These yeast studies will help guide the analysis of novel human "hMLH1-or hPMS1-interacting" proteins in much the same way that the yeast studies on PMS1 and MLH1 have influenced our studies of the human MLH1 and PMS1 genes.

Production of Antibodies

By using our knowledge of the DNA sequences for hMLH1 and hPMS1, we can synthesize all or portions of the predicted protein structures for the purpose of producing antibodies. One important use for antibodies directed to hMLH1 and hPMS1 proteins will be for capturing other proteins which may be involved in DNA mismatch repair. For example, by employing coimmuno-precipitation techniques, antibodies directed to either hMLH1 or hPMS1 may be precipitated along with other associated proteins which are functionally and/or physically related. Another important use for antibodies will be for the purpose of isolating hMLH1 and hPMS1 proteins from tumor tissue. The hMLH1 and hPMS1 proteins from tumors can then be characterized for the purpose of determining appropriate treatment strategies.

We are in the process of developing monoclonal antibodies directed to the hMLH1 protein. We have used the following procedure to produce polyclonal antibodies directed to the human and mouse forms of PMS1 protein.

We inserted a 3' fragment of the mouse PMS1 cDNA in the bacterial expression plasmid vector, pET (Novagen, Madison, Wis.). The expected expressed portion of the mouse PMS1 protein corresponds to a region of approximately 200 amino acids at the end of the PMS1 protein. This portion of the mPMS1 is conserved with yeast PMS1 but is not conserved with either the human or the mouse MLH1 proteins. One reason that we selected this portion of the PMS1 protein for producing antibodies is that we did not want the resulting antibodies to cross-react with MLH1. The mouse PMS1 protein fragment was highly expressed in E. coli, purified from a polyacrylamide gel and the eluted protein was then prepared for animal injections. Approximately 2 mg of the PMS1 protein fragment was sent to the Pocono Rabbit Farm (Pa.) for injections into rabbits. Sera from rabbits multiple times was tittered against the PMS1 antigen using standard ELISA techniques. Rabbit antibodies specific to mouse PMS1 protein were affinity-purified using columns containing immobilized mouse PMS1 protein. The affinity-purified polyclonal antibody preparation was tested further using Western blotting and dot blotting. We found that the polyclonal antibodies recognized, not only the mouse PMS1 protein, but also the human PMS1 protein which is very similar. Based upon the Western blots, there is no indication that other proteins were recognized strongly by our antibody, including either the human or mouse MLH1 proteins.

Mouse MLH1

Using the procedure outlined above with reference to FIG. 1, we have determined a partial nucleotide sequence of mouse MLH1 cDNA, as shown in FIG. 13. FIG. 14 shows the corresponding predicted amino acid sequence for mMLH1 protein in comparison to the predicted hMLH1 protein sequence. Comparison of the mouse and human MLH1 proteins as well as the comparison of hMLH1 with yeast MLH1 proteins, as shown in FIG. 9, indicate a high degree of conservation.

Mouse PMS1

Using the procedures discussed above with reference to FIG. 1, we isolated and sequenced the mouse PMS1 gene, as shown in FIG. 15. This cDNA sequence encodes a predicted protein of 864 amino acids, as shown in FIG. 16, where it is compared to the predicted amino acid sequence for hPMS1. The degree of identity between the predicted mouse and human PMS1 proteins is high, as would be expected between two mammals. Similarly, as noted above, there is strong similarity between the human PMS1 protein and the yeast DNA mismatch repair protein PMS1, as shown in FIG. 11. The fact that yeast PMS1 and MLH1 function in yeast to repair DNA mismatches, strongly suggests that human and mice PMS1 and MLH1 are also mismatch repair proteins.

Uses for Mouse MLH1 and PMS1

We believe our isolation and characterization of mMLH1 and mPMS1 genes will have many research applications. For example, as already discussed above, we have used our knowledge of the mPMS1 gene to produce antibodies which react specifically with hPMS1. We have already explained that antibodies directed to the human proteins, MLH1 or PMS1 may be used for both research purposes as well as diagnostic purposes.

We also believe that our knowledge of mPMS1 and mMLH1 will be useful for constructing mouse models in order to study the consequences of DNA mismatch repair defects. We expect that mPMS1 or mMLH1 defective mice will be highly prone to cancer because chromosome 2p and 3p-associated HNPCC are each due to a defect in a mismatch repair gene.¹,2 As noted above, we have already produced chimeric mice which carry an mPMS1 defective gene. We are currently constructing mice heterozygous for mPMS1 or mMLH1 mutation. These heterozygous mice should provide useful animal models for studying human cancer, in particular HNPCC. The mice will be useful for analysis of both intrinsic and extrinsic factors that determine cancer risk and progression. Also, cancers associated with mismatch repair deficiency may respond differently to conventional therapy in comparison to other cancers. Such animal models will be useful for determining if differences exist, and allow the development of regimes for the effective treatment of these types of tumors. Such animal models may also be used to study the relationship between hereditary versus dietary factors in carcinogenesis.

    __________________________________________________________________________     #             SEQUENCE LISTING     - (1) GENERAL INFORMATION:     -    (iii) NUMBER OF SEQUENCES: 30     - (2) INFORMATION FOR SEQ ID NO:1:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 29 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #ID NO:1: (xi) SEQUENCE DESCRIPTION: SEQ     #            29    CNCC NCKRAANCC     - (2) INFORMATION FOR SEQ ID NO:2:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 29 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     #ID NO:2: (xi) SEQUENCE DESCRIPTION: SEQ     #            29    ARYT NGTNGANAA     - (2) INFORMATION FOR SEQ ID NO:3:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 15 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA (genomic)     #ID NO:3: (xi) SEQUENCE DESCRIPTION: SEQ     #    15     - (2) INFORMATION FOR SEQ ID NO:4:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 15 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA (genomic)     #ID NO:4: (xi) SEQUENCE DESCRIPTION: SEQ     #    15     - (2) INFORMATION FOR SEQ ID NO:5:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 361 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:5: (xi) SEQUENCE DESCRIPTION: SEQ     -      Met Pro Ile Gln Val Leu Pro Pro - # Gln Leu Ala Asn Gln Ile Ala     Ala     #   15     -      Gly Glu Val Val Glu Arg Pro Ala - # Ser Val Val Lys Glu Leu Val     Glu     #                 30     -      Asn Ser Leu Asp Ala Gly Ala Thr - # Arg Val Asp Ile Asp Ile Glu     Arg     #             45     -      Gly Gly Ala Lys Leu Ile Arg Ile - # Arg Asp Asn Gly Cys Gly Ile     Lys     #         60     -      Lys Glu Glu Leu Ala Leu Ala Leu - # Ala Arg His Ala Thr Ser Lys     Ile     #     80     -      Ala Ser Leu Asp Asp Leu Glu Ala - # Ile Ile Ser Leu Gly Phe Arg     Gly     #   95     -      Glu Ala Leu Ala Ser Ile Ser Ser - # Val Ser Arg Leu Thr Leu Thr     Ser     #                110     -      Arg Thr Ala Glu Gln Ala Glu Ala - # Trp Gln Ala Tyr Ala Glu Gly     Arg     #            125     -      Asp Met Asp Val Thr Val Lys Pro - # Ala Ala His Pro Val Gly Thr     Thr     #        140     -      Leu Glu Val Leu Asp Leu Phe Tyr - # Asn Thr Pro Ala Arg Arg Lys     Phe     #    160     -      Met Arg Thr Glu Lys Thr Glu Phe - # Asn His Ile Asp Glu Ile Ile     Arg     #   175     -      Arg Ile Ala Leu Ala Arg Phe Asp - # Val Thr Leu Asn Leu Ser His     Asn     #                190     -      Gly Lys Leu Val Arg Gln Tyr Arg - # Ala Val Ala Lys Asp Gly Gln     Lys     #            205     -      Glu Arg Arg Leu Gly Ala Ile Cys - # Gly Thr Pro Phe Leu Glu Gln     Ala     #        220     -      Leu Ala Ile Glu Trp Gln His Gly - # Asp Lys Thr Lys Arg Gly Trp     Val     #    240     -      Ala Asp Pro Asn His Thr Thr Thr - # Ala Leu Thr Glu Ile Gln Tyr     Cys     #   255     -      Tyr Val Asn Gly Arg Met Met Arg - # Asp Arg Leu Ile Asn His Ala     Ile     #                270     -      Arg Gln Ala Cys Glu Asp Lys Leu - # Gly Ala Asp Gln Gln Pro Ala     Phe     #            285     -      Val Leu Tyr Leu Glu Ile Asp Pro - # His Gln Val Asp Val Asn Val     His     #        300     -      Pro Ala Lys His Glu Val Arg Phe - # His Gln Ser Arg Leu Val His     Asp     #    320     -      Phe Ile Tyr Gln Gly Val Leu Ser - # Val Leu Gln Gln Gln Thr Glu     Thr     #   335     -      Ala Leu Pro Leu Glu Glu Ile Ala - # Pro Ala Pro Arg His Val Gln     Glu     #                350     -      Asn Arg Ile Ala Ala Gly Arg Asn - # His     #            360     - (2) INFORMATION FOR SEQ ID NO:6:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 538 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:6: (xi) SEQUENCE DESCRIPTION: SEQ     -      Met Ser His Ile Ile Glu Leu Pro - # Glu Met Leu Ala Asn Gln Ile     Ala     #   15     -      Ala Gly Glu Val Ile Glu Arg Pro - # Ala Ser Val Cys Lys Glu Leu     Val     #                 30     -      Glu Asn Ala Ile Asp Ala Gly Ser - # Ser Gln Ile Ile Ile Glu Ile     Glu     #             45     -      Glu Ala Gly Leu Lys Lys Val Gln - # Ile Thr Asp Asn Gly His Gly     Ile     #         60     -      Ala His Asp Glu Val Glu Leu Ala - # Leu Arg Arg His Ala Thr Ser     Lys     #     80     -      Ile Lys Asn Gln Ala Asp Leu Phe - # Arg Ile Arg Thr Leu Gly Phe     Arg     #   95     -      Gly Glu Ala Leu Pro Ser Ile Ala - # Ser Val Ser Val Leu Thr Leu     Leu     #                110     -      Thr Ala Val Asp Gly Ala Ser His - # Gly Thr Lys Leu Val Ala Arg     Gly     #            125     -      Gly Glu Val Glu Glu Val Ile Pro - # Ala Thr Ser Pro Val Gly Thr     Lys     #        140     -      Val Cys Val Glu Asp Leu Phe Phe - # Asn Thr Pro Ala Arg Leu Lys     Tyr     #    160     -      Met Lys Ser Gln Gln Ala Glu Leu - # Ser His Ile Ile Asp Ile Val     Asn     #   175     -      Arg Leu Gly Leu Ala His Pro Glu - # Ile Ser Phe Ser Leu Ile Ser     Asp     #                190     -      Gly Lys Glu Met Thr Arg Thr Ala - # Gly Thr Gly Gln Leu Arg Gln     Ala     #            205     -      Ile Ala Gly Ile Tyr Gly Leu Val - # Ser Ala Lys Lys Met Ile Glu     Ile     #        220     -      Glu Asn Ser Asp Leu Asp Phe Glu - # Ile Ser Gly Phe Val Ser Leu     Pro     #    240     -      Glu Leu Thr Arg Ala Asn Arg Asn - # Tyr Ile Ser Leu Phe Ile Asn     Gly     #   255     -      Arg Tyr Ile Lys Asn Phe Leu Leu - # Asn Arg Ala Ile Leu Asp Gly     Phe     #                270     -      Gly Ser Lys Leu Met Val Gly Arg - # Phe Pro Leu Ala Val Ile His     Ile     #            285     -      His Ile Asp Pro Tyr Leu Ala Asp - # Val Asn Val His Pro Thr Lys     Gln     #        300     -      Glu Val Arg Ile Ser Lys Glu Lys - # Glu Leu Met Thr Leu Val Ser     Glu     #    320     -      Ala Ile Ala Asn Ser Leu Lys Glu - # Gln Thr Leu Ile Pro Asp Ala     Leu     #   335     -      Glu Asn Leu Ala Lys Ser Thr Val - # Arg Asn Arg Glu Lys Val Glu     Gln     #                350     -      Thr Ile Leu Pro Leu Ser Phe Pro - # Glu Leu Glu Phe Phe Gly Gln     Met     #            365     -      His Gly Thr Tyr Leu Phe Ala Gln - # Gly Arg Asp Gly Leu Tyr Ile     Ile     #        380     -      Asp Gln His Ala Ala Gln Glu Arg - # Val Lys Tyr Glu Glu Tyr Arg     Glu     #    400     -      Ser Ile Gly Asn Val Asp Gln Ser - # Gln Gln Gln Leu Leu Val Pro     Tyr     #   415     -      Ile Phe Glu Phe Pro Ala Asp Asp - # Ala Leu Arg Leu Lys Glu Arg     Met     #                430     -      Pro Leu Leu Glu Glu Val Gly Val - # Phe Leu Ala Glu Tyr Gly Glu     Asn     #            445     -      Gln Phe Ile Leu Arg Glu His Pro - # Ile Trp Met Ala Glu Glu Glu     Ile     #        460     -      Glu Ser Gly Ile Tyr Glu Met Cys - # Asp Met Leu Leu Leu Thr Lys     Glu     #    480     -      Val Ser Ile Lys Lys Tyr Arg Ala - # Glu Leu Ala Ile Met Met Ser     Cys     #   495     -      Lys Arg Ser Ile Lys Ala Asn His - # Arg Ile Asp Asp His Ser Ala     Arg     #                510     -      Gln Leu Leu Tyr Gln Leu Ser Gln - # Cys Asp Asn Pro Tyr Asn Cys     Pro     #            525     -      His Gly Arg Pro Val Leu Val His - # Phe Thr     #        535     - (2) INFORMATION FOR SEQ ID NO:7:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 607 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:7: (xi) SEQUENCE DESCRIPTION: SEQ     -      Met Phe His His Ile Glu Asn Leu - # Leu Ile Glu Thr Glu Lys Arg     Cys     #   15     -      Lys Gln Lys Glu Gln Arg Tyr Ile - # Pro Val Lys Tyr Leu Phe Ser     Met     #                 30     -      Thr Gln Ile His Gln Ile Asn Asp - # Ile Asp Val His Arg Ile Thr     Ser     #             45     -      Gly Gln Val Ile Thr Asp Leu Thr - # Thr Ala Val Lys Glu Leu Val     Asp     #         60     -      Asn Ser Ile Asp Ala Asn Ala Asn - # Gln Ile Glu Ile Ile Phe Lys     Asp     #     80     -      Tyr Gly Leu Glu Ser Ile Glu Cys - # Ser Asp Asn Gly Asp Gly Ile     Asp     #   95     -      Pro Ser Asn Tyr Glu Phe Leu Ala - # Leu Lys His Tyr Thr Ser Lys     Ile     #                110     -      Ala Lys Phe Gln Asp Val Ala Lys - # Val Gln Thr Leu Gly Phe Arg     Gly     #            125     -      Glu Ala Leu Ser Ser Leu Cys Gly - # Ile Ala Lys Leu Ser Val Ile     Thr     #        140     -      Thr Thr Ser Pro Pro Lys Ala Asp - # Lys Glu Leu Tyr Asp Met Val     Gly     #    160     -      His Ile Thr Ser Lys Thr Thr Thr - # Ser Arg Asn Lys Gly Thr Thr     Val     #   175     -      Leu Val Ser Gln Leu Phe His Asn - # Leu Pro Val Arg Gln Lys Glu     Phe     #                190     -      Ser Lys Thr Phe Lys Arg Gln Phe - # Thr Lys Cys Leu Thr Val Ile     Gln     #            205     -      Gly Tyr Ala Ile Ile Asn Ala Ala - # Ile Lys Phe Ser Val Trp Asn     Ile     #        220     -      Thr Pro Lys Gly Lys Lys Asn Leu - # Ile Leu Ser Thr Met Arg Asn     Ser     #    240     -      Ser Met Arg Lys Asn Ile Ser Ser - # Val Phe Gly Ala Gly Gly Met     Arg     #   255     -      Gly Glu Leu Glu Val Asp Leu Val - # Leu Asp Leu Asn Pro Phe Lys     Asn     #                270     -      Arg Met Leu Gly Lys Tyr Thr Asp - # Asp Pro Asp Phe Leu Asp Leu     Asp     #            285     -      Tyr Lys Ile Arg Val Lys Gly Tyr - # Ile Ser Gln Asn Ser Phe Gly     Cys     #        300     -      Gly Arg Asn Ser Lys Asp Arg Gln - # Phe Ile Tyr Val Asn Lys Arg     Pro     #    320     -      Val Glu Tyr Ser Thr Leu Leu Lys - # Cys Cys Asn Glu Val Tyr Lys     Thr     #   335     -      Phe Asn Asn Val Gln Phe Pro Ala - # Val Phe Leu Asn Leu Glu Leu     Pro     #                350     -      Met Ser Leu Ile Asp Val Asn Val - # Thr Pro Asp Lys Arg Val Ile     Leu     #            365     -      Leu His Asn Glu Arg Ala Val Ile - # Asp Ile Phe Lys Thr Thr Leu     Ser     #        380     -      Asp Tyr Tyr Asn Arg Gln Glu Leu - # Ala Leu Pro Lys Arg Met Cys     Ser     #    400     -      Gln Ser Glu Gln Gln Ala Gln Lys - # Arg Leu Leu Thr Glu Val Phe     Asp     #   415     -      Asp Asp Phe Lys Lys Met Glu Val - # Val Gly Gln Phe Asn Leu Gly     Phe     #                430     -      Ile Ile Val Thr Arg Lys Val Asp - # Asn Lys Ser Asp Leu Phe Ile     Val     #            445     -      Asp Gln His Ala Ser Asp Glu Lys - # Tyr Asn Phe Glu Thr Leu Gln     Ala     #        460     -      Val Thr Val Phe Lys Ser Gln Lys - # Leu Ile Ile Pro Gln Pro Val     Glu     #    480     -      Leu Ser Val Ile Asp Glu Leu Val - # Val Leu Asp Asn Leu Pro Val     Phe     #   495     -      Glu Lys Asn Gly Phe Lys Leu Lys - # Ile Asp Glu Glu Glu Glu Phe     Gly     #                510     -      Ser Arg Val Lys Leu Leu Ser Leu - # Pro Thr Ser Lys Gln Thr Leu     Phe     #            525     -      Asp Leu Gly Asp Phe Asn Glu Leu - # Ile His Leu Ile Lys Glu Asp     Gly     #        540     -      Gly Leu Arg Arg Asp Asn Ile Arg - # Cys Ser Lys Ile Arg Ser Met     Phe     #    560     -      Ala Met Arg Ala Cys Arg Ser Ser - # Ile Met Ile Gly Lys Pro Leu     Asn     #   575     -      Lys Lys Thr Met Thr Arg Val Val - # His Asn Leu Ser Glu Leu Asp     Lys     #                590     -      Pro Trp Asn Cys Pro His Gly Arg - # Pro Thr Met Arg His Leu Met     #            605     - (2) INFORMATION FOR SEQ ID NO:8:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 2484 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     -   (viii) POSITION IN GENOME:               (B) MAP POSITION: 3p21. - #3-23     #ID NO:8: (xi) SEQUENCE DESCRIPTION: SEQ     - CTTGGCTCTT CTGGCGCCAA AATGTCGTTC GTGGCAGGGG TTATTCGGCG GC - #TGGACGAG       60     - ACAGTGGTGA ACCGCATCGC GGCGGGGGAA GTTATCCAGC GGCCAGCTAA TG - #CTATCAAA      120     - GAGATGATTG AGAACTGTTT AGATGCAAAA TCCACAAGTA TTCAAGTGAT TG - #TTAAAGAG      180     - GGAGGCCTGA AGTTGATTCA GATCCAAGAC AATGGCACCG GGATCAGGAA AG - #AAGATCTG      240     - GATATTGTAT GTGAAAGGTT CACTACTAGT AAACTGCAGT CCTTTGAGGA TT - #TAGCCAGT      300     - ATTTCTACCT ATGGCTTTCG AGGTGAGGCT TTGGCCAGCA TAAGCCATGT GG - #CTCATGTT      360     - ACTATTACAA CGAAAACAGC TGATGGAAAG TGTGCATACA GAGCAAGTTA CT - #CAGATGGA      420     - AAACTGAAAG CCCCTCCTAA ACCATGTGCT GGCAATCAAG GGACCCAGAT CA - #CGGTGGAG      480     - GACCTTTTTT ACAACATAGC CACGAGGAGA AAAGCTTTAA AAAATCCAAG TG - #AAGAATAT      540     - GGGAAAATTT TGGAAGTTGT TGGCAGGTAT TCAGTACACA ATGCAGGCAT TA - #GTTTCTCA      600     - GTTAAAAAAC AAGGAGAGAC AGTAGCTGAT GTTAGGACAC TACCCAATGC CT - #CAACCGTG      660     - GACAATATTC GCTCCATCTT TGGAAATGCT GTTAGTCGAG AACTGATAGA AA - #TTGGATGT      720     - GAGGATAAAA CCCTAGCCTT CAAAATGAAT GGTTACATAT CCAATGCAAA CT - #ACTCAGTG      780     - AAGAAGTGCA TCTTCTTACT CTTCATCAAC CATCGTCTGG TAGAATCAAC TT - #CCTTGAGA      840     - AAAGCCATAG AAACAGTGTA TGCAGCCTAT TTGCCCAAAA ACACACACCC AT - #TCCTGTAC      900     - CTCAGTTTAG AAATCAGTCC CCAGAATGTG GATGTTAATG TGCACCCCAC AA - #AGCATGAA      960     - GTTCACTTCC TGCACGAGGA GAGCATCCTG GAGCGGGTGC AGCAGCACAT CG - #AGAGCAAG     1020     - CTCCTGGGCT CCAATTCCTC CAGGATGTAC TTCACCCAGA CTTTGCTACC AG - #GACTTGCT     1080     - GGCCCCTCTG GGGAGATGGT TAAATCCACA ACAAGTCTGA CCTCGTCTTC TA - #CTTCTGGA     1140     - AGTAGTGATA AGGTCTATGC CCACCAGATG GTTCGTACAG ATTCCCGGGA AC - #AGAAGCTT     1200     - GATGCATTTC TGCAGCCTCT GAGCAAACCC CTGTCCAGTC AGCCCCAGGC CA - #TTGTCACA     1260     - GAGGATAAGA CAGATATTTC TAGTGGCAGG GCTAGGCAGC AAGATGAGGA GA - #TGCTTGAA     1320     - CTCCCAGCCC CTGCTGAAGT GGCTGCCAAA AATCAGAGCT TGGAGGGGGA TA - #CAACAAAG     1380     - GGGACTTCAG AAATGTCAGA GAAGAGAGGA CCTACTTCCA GCAACCCCAG AA - #AGAGACAT     1440     - CGGGAAGATT CTGATGTGGA AATGGTGGAA GATGATTCCC GAAAGGAAAT GA - #CTGCAGCT     1500     - TGTACCCCCC GGAGAAGGAT CATTAACCTC ACTAGTGTTT TGAGTCTCCA GG - #AAGAAATT     1560     - AATGAGCAGG GACATGAGGT TCTCCGGGAG ATGTTGCATA ACCACTCCTT CG - #TGGGCTGT     1620     - GTGAATCCTC AGTGGGCCTT GGCACAGCAT CAAACCAAGT TATACCTTCT CA - #ACACCACC     1680     - AAGCTTAGTG AAGAACTGTT CTACCAGATA CTCATTTATG ATTTTGCCAA TT - #TTGGTGTT     1740     - CTCAGGTTAT CGGAGCCAGC ACCGCTCTTT GACCTTGCCA TGCTTGCCTT AG - #ATAGTCCA     1800     - GAGAGTGGCT GGACAGAGGA AGATGGTCCC AAAGAAGGAC TTGCTGAATA CA - #TTGTTGAG     1860     - TTTCTGAAGA AGAAGGCTGA GATGCTTGCA GACTATTTCT CTTTGGAAAT TG - #ATGAGGAA     1920     - GGGAACCTGA TTGGATTACC CCTTCTGATT GACAACTATG TGCCCCCTTT GG - #AGGGACTG     1980     - CCTATCTTCA TTCTTCGACT AGCCACTGAG GTGAATTGGG ACGAAGAAAA GG - #AATGTTTT     2040     - GAAAGCCTCA GTAAAGAATG CGCTATGTTC TATTCCATCC GGAAGCAGTA CA - #TATCTGAG     2100     - GAGTCGACCC TCTCAGGCCA GCAGAGTGAA GTGCCTGGCT CCATTCCAAA CT - #CCTGGAAG     2160     - TGGACTGTGG AACACATTGT CTATAAAGCC TTGCGCTCAC ACATTCTGCC TC - #CTAAACAT     2220     - TTCACAGAAG ATGGAAATAT CCTGCAGCTT GCTAACCTGC CTGATCTATA CA - #AAGTCTTT     2280     - GAGAGGTGTT AAATATGGTT ATTTATGCAC TGTGGGATGT GTTCTTCTTT CT - #CTGTATTC     2340     - CGATACAAAG TGTTGTATCA AAGTGTGATA TACAAAGTGT ACCAACATAA GT - #GTTGGTAG     2400     - CACTTAAGAC TTATACTTGC CTTCTGATAG TATTCCTTTA TACACAGTGG AT - #TGATTATA     2460     #              2484TTAA CATA     - (2) INFORMATION FOR SEQ ID NO:9:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 756 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:9: (xi) SEQUENCE DESCRIPTION: SEQ     -      Met Ser Phe Val Ala Gly Val Ile - # Arg Arg Leu Asp Glu Thr Val     Val     #   15     -      Asn Arg Ile Ala Ala Gly Glu Val - # Ile Gln Arg Pro Ala Asn Ala     Ile     #                 30     -      Lys Glu Met Ile Glu Asn Cys Leu - # Asp Ala Lys Ser Thr Ser Ile     Gln     #             45     -      Val Ile Val Lys Glu Gly Gly Leu - # Lys Leu Ile Gln Ile Gln Asp     Asn     #         60     -      Gly Thr Gly Ile Arg Lys Glu Asp - # Leu Asp Ile Val Cys Glu Arg     Phe     #     80     -      Thr Thr Ser Lys Leu Gln Ser Phe - # Glu Asp Leu Ala Ser Ile Ser     Thr     #   95     -      Tyr Gly Phe Arg Gly Glu Ala Leu - # Ala Ser Ile Ser His Val Ala     His     #                110     -      Val Thr Ile Thr Thr Lys Thr Ala - # Asp Gly Lys Cys Ala Tyr Arg     Ala     #            125     -      Ser Tyr Ser Asp Gly Lys Leu Lys - # Ala Pro Pro Lys Pro Cys Ala     Gly     #        140     -      Asn Gln Gly Thr Gln Ile Thr Val - # Glu Asp Leu Phe Tyr Asn Ile     Ala     #    160     -      Thr Arg Arg Lys Ala Leu Lys Asn - # Pro Ser Glu Glu Tyr Gly Lys     Ile     #   175     -      Leu Glu Val Val Gly Arg Tyr Ser - # Val His Asn Ala Gly Ile Ser     Phe     #                190     -      Ser Val Lys Lys Gln Gly Glu Thr - # Val Ala Asp Val Arg Thr Leu     Pro     #            205     -      Asn Ala Ser Thr Val Asp Asn Ile - # Arg Ser Ile Phe Gly Asn Ala     Val     #        220     -      Ser Arg Glu Leu Ile Glu Ile Gly - # Cys Glu Asp Lys Thr Leu Ala     Phe     #    240     -      Lys Met Asn Gly Tyr Ile Ser Asn - # Ala Asn Tyr Ser Val Lys Lys     Cys     #   255     -      Ile Phe Leu Leu Phe Ile Asn His - # Arg Leu Val Glu Ser Thr Ser     Leu     #                270     -      Arg Lys Ala Ile Glu Thr Val Tyr - # Ala Ala Tyr Leu Pro Lys Asn     Thr     #            285     -      His Pro Phe Leu Tyr Leu Ser Leu - # Glu Ile Ser Pro Gln Asn Val     Asp     #        300     -      Val Asn Val His Pro Thr Lys His - # Glu Val His Phe Leu His Glu     Glu     #    320     -      Ser Ile Leu Glu Arg Val Gln Gln - # His Ile Glu Ser Lys Leu Leu     Gly     #   335     -      Ser Asn Ser Ser Arg Met Tyr Phe - # Thr Gln Thr Leu Leu Pro Gly     Leu     #                350     -      Ala Gly Pro Ser Gly Glu Met Val - # Lys Ser Thr Thr Ser Leu Thr     Ser     #            365     -      Ser Ser Thr Ser Gly Ser Ser Asp - # Lys Val Tyr Ala His Gln Met     Val     #        380     -      Arg Thr Asp Ser Arg Glu Gln Lys - # Leu Asp Ala Phe Leu Gln Pro     Leu     #    400     -      Ser Lys Pro Leu Ser Ser Gln Pro - # Gln Ala Ile Val Thr Glu Asp     Lys     #   415     -      Thr Asp Ile Ser Ser Gly Arg Ala - # Arg Gln Gln Asp Glu Glu Met     Leu     #                430     -      Glu Leu Pro Ala Pro Ala Glu Val - # Ala Ala Lys Asn Gln Ser Leu     Glu     #            445     -      Gly Asp Thr Thr Lys Gly Thr Ser - # Glu Met Ser Glu Lys Arg Gly     Pro     #        460     -      Thr Ser Ser Asn Pro Arg Lys Arg - # His Arg Glu Asp Ser Asp Val     Glu     #    480     -      Met Val Glu Asp Asp Ser Arg Lys - # Glu Met Thr Ala Ala Cys Thr     Pro     #   495     -      Arg Arg Arg Ile Ile Asn Leu Thr - # Ser Val Leu Ser Leu Gln Glu     Glu     #                510     -      Ile Asn Glu Gln Gly His Glu Val - # Leu Arg Glu Met Leu His Asn     His     #            525     -      Ser Phe Val Gly Cys Val Asn Pro - # Gln Trp Ala Leu Ala Gln His     Gln     #        540     -      Thr Lys Leu Tyr Leu Leu Asn Thr - # Thr Lys Leu Ser Glu Glu Leu     Phe     #    560     -      Tyr Gln Ile Leu Ile Tyr Asp Phe - # Ala Asn Phe Gly Val Leu Arg     Leu     #   575     -      Ser Glu Pro Ala Pro Leu Phe Asp - # Leu Ala Met Leu Ala Leu Asp     Ser     #                590     -      Pro Glu Ser Gly Trp Thr Glu Glu - # Asp Gly Pro Lys Glu Gly Leu     Ala     #            605     -      Glu Tyr Ile Val Glu Phe Leu Lys - # Lys Lys Ala Glu Met Leu Ala     Asp     #        620     -      Tyr Phe Ser Leu Glu Ile Asp Glu - # Glu Gly Asn Leu Ile Gly Leu     Pro     #    640     -      Leu Leu Ile Asp Asn Tyr Val Pro - # Pro Leu Glu Gly Leu Pro Ile     Phe     #   655     -      Ile Leu Arg Leu Ala Thr Glu Val - # Asn Trp Asp Glu Glu Lys Glu     Cys     #                670     -      Phe Glu Ser Leu Ser Lys Glu Cys - # Ala Met Phe Tyr Ser Ile Arg     Lys     #            685     -      Gln Tyr Ile Ser Glu Glu Ser Thr - # Leu Ser Gly Gln Gln Ser Glu     Val     #        700     -      Pro Gly Ser Ile Pro Asn Ser Trp - # Lys Trp Thr Val Glu His Ile     Val     #    720     -      Tyr Lys Ala Leu Arg Ser His Ile - # Leu Pro Pro Lys His Phe Thr     Glu     #   735     -      Asp Gly Asn Ile Leu Gln Leu Ala - # Asn Leu Pro Asp Leu Tyr Lys     Val     #                750     -      Phe Glu Arg Cys                  755     - (2) INFORMATION FOR SEQ ID NO:10:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 361 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     -   (viii) POSITION IN GENOME:               (B) MAP POSITION: 3p21. - #3-23     #ID NO:10:(xi) SEQUENCE DESCRIPTION: SEQ     - TTAATGAGGC ACTATTGTTT GTATTTGGAG TTTGTTATCA TTGCTTGGCT CA - #TATTAAAA       60     - TATGTACATT AGAGTAGTTG CAGACTGATA AATTATTTTC TGTTTGATTT GC - #CAGTTTAG      120     - ATGCAAAATC CACAAGTATT CAAGTGATTG TTAAAGAGGG AGGCCTGAAG TT - #GATTCAGA      180     - TCCAAGACAA TGGCACCGGG ATCAGGGTAA GTAAAACCTC AAAGTAGCAG GA - #TGTTTGTG      240     - CGCTTCATGG AAGAGTCAGG ACCTTTCTCT GTTCTGGAAA CTAGGCTTTT GC - #AGATGGGA      300     - TTTTTTCACT GAAAAATTCA ACACCAACAA TAAATATTTA TTGAGTACCT AT - #TATTTGCG      360     #              361     - (2) INFORMATION FOR SEQ ID NO:11:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 341 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     -   (viii) POSITION IN GENOME:               (B) MAP POSITION: 3p21. - #3-23     #ID NO:11:(xi) SEQUENCE DESCRIPTION: SEQ     -      Gly Ala Ala Thr Thr Cys Ala Ala - # Ala Gly Ala Gly Ala Thr Thr     Thr     #   15     -      Gly Gly Ala Ala Ala Ala Thr Gly - # Ala Gly Thr Ala Ala Cys Ala     Thr     #                 30     -      Gly Ala Thr Thr Ala Thr Thr Thr - # Ala Cys Thr Cys Ala Thr Cys     Thr     #             45     -      Thr Thr Thr Thr Gly Gly Thr Ala - # Thr Cys Thr Ala Ala Cys Ala     Ala     #         60     -      Ala Ala Gly Ala Ala Gly Ala Thr - # Cys Thr Gly Gly Ala Thr Ala     Thr     #     80     -      Thr Gly Thr Ala Thr Gly Thr Gly - # Ala Ala Ala Gly Gly Lys Thr     Cys     #   95     -      Ala Cys Thr Ala Cys Thr Ala Gly - # Thr Ala Ala Ala Cys Thr Gly     Cys     #                110     -      Ala Gly Thr Cys Cys Thr Thr Thr - # Gly Ala Gly Gly Ala Asp Thr     Thr     #            125     -      Thr Ala Gly Cys Cys Ala Gly Thr - # Ala Thr Thr Thr Cys Thr Ala     Cys     #        140     -      Cys Thr Ala Thr Gly Gly Cys Thr - # Thr Thr Cys Gly Ala Gly Gly     Thr     #    160     -      Gly Ala Gly Gly Thr Ala Ala Gly - # Cys Thr Ala Ala Ala Gly Ala     Thr     #   175     -      Thr Cys Ala Ala Gly Ala Ala Ala - # Thr Gly Thr Lys Thr Ala Ala     Ala     #                190     -      Ala Thr Ala Thr Cys Cys Thr Cys - # Cys Thr Gly Thr Gly Ala Thr     Gly     #            205     -      Ala Cys Ala Thr Thr Gly Thr Tyr - # Thr Gly Thr Cys Ala Thr Thr     Thr     #        220     -      Gly Thr Thr Ala Gly Thr Ala Thr - # Gly Thr Ala Thr Thr Thr Cys     Thr     #    240     -      Cys Ala Ala Cys Ala Thr Ala Gly - # Ala Thr Ala Ala Ala Thr Ala     Ala     #   255     -      Gly Gly Thr Thr Thr Gly Gly Thr - # Ala Cys Cys Thr Thr Thr Thr     Ala     #                270     -      Cys Thr Thr Gly Thr Thr Ala Ala - # Ala Thr Gly Thr Ala Thr Gly     Cys     #            285     -      Ala Ala Ala Thr Gly Thr Ala Thr - # Gly Cys Ala Ala Ala Thr Cys     Thr     #        300     -      Gly His Gly Cys Ala Ala Ala Cys - # Thr Thr Ala Ala Thr Gly Ala     Asp     #    320     -      Cys Thr Thr Thr Ala Ala Cys Thr - # Thr Thr Cys Ala Ala Ala Gly     Ala     #   335     -      Cys Thr Gly Ala Gly                      340     - (2) INFORMATION FOR SEQ ID NO:12:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 756 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:12:(xi) SEQUENCE DESCRIPTION: SEQ     -      Met Ser Phe Val Ala Gly Val Ile - # Arg Arg Leu Asp Glu Thr Val     Val     #   15     -      Asn Arg Ile Ala Ala Gly Glu Val - # Ile Gln Arg Pro Ala Asn Ala     Ile     #                 30     -      Lys Glu Met Ile Glu Asn Cys Leu - # Asp Ala Lys Ser Thr Ser Ile     Gln     #             45     -      Val Ile Val Lys Glu Gly Gly Leu - # Lys Leu Ile Gln Ile Gln Asp     Asn     #         60     -      Gly Thr Gly Ile Arg Lys Glu Asp - # Leu Asp Ile Val Cys Glu Arg     Phe     #     80     -      Thr Thr Ser Lys Leu Gln Ser Phe - # Glu Asp Leu Ala Ser Ile Ser     Thr     #   95     -      Tyr Gly Phe Arg Gly Glu Ala Leu - # Ala Ser Ile Ser His Val Ala     His     #                110     -      Val Thr Ile Thr Thr Lys Thr Ala - # Asp Gly Lys Cys Ala Tyr Arg     Ala     #            125     -      Ser Tyr Ser Asp Gly Lys Leu Lys - # Ala Pro Pro Lys Pro Cys Ala     Gly     #        140     -      Asn Gln Gly Thr Gln Ile Thr Val - # Glu Asp Leu Phe Tyr Asn Ile     Ala     #    160     -      Thr Arg Arg Lys Ala Leu Lys Asn - # Pro Ser Glu Glu Tyr Gly Lys     Ile     #   175     -      Leu Glu Val Val Gly Arg Tyr Ser - # Val His Asn Ala Gly Ile Ser     Phe     #                190     -      Ser Val Lys Lys Gln Gly Glu Thr - # Val Ala Asp Val Arg Thr Leu     Pro     #            205     -      Asn Ala Ser Thr Val Asp Asn Ile - # Arg Ser Ile Phe Gly Asn Ala     Val     #        220     -      Ser Arg Glu Leu Ile Glu Ile Gly - # Cys Glu Asp Lys Thr Leu Ala     Phe     #    240     -      Lys Met Asn Gly Tyr Ile Ser Asn - # Ala Asn Tyr Ser Val Lys Lys     Cys     #   255     -      Ile Phe Leu Leu Phe Ile Asn His - # Arg Leu Val Glu Ser Thr Ser     Leu     #                270     -      Arg Lys Ala Ile Glu Thr Val Tyr - # Ala Ala Tyr Leu Pro Lys Asn     Thr     #            285     -      His Pro Phe Leu Tyr Leu Ser Leu - # Glu Ile Ser Pro Gln Asn Val     Asp     #        300     -      Val Asn Val His Pro Thr Lys His - # Glu Val His Phe Leu His Glu     Glu     #    320     -      Ser Ile Leu Glu Arg Val Gln Gln - # His Ile Glu Ser Lys Leu Leu     Gly     #   335     -      Ser Asn Ser Ser Arg Met Tyr Phe - # Thr Gln Thr Leu Leu Pro Gly     Leu     #                350     -      Ala Gly Pro Ser Gly Glu Met Val - # Lys Ser Thr Thr Ser Leu Thr     Ser     #            365     -      Ser Ser Thr Ser Gly Ser Ser Asp - # Lys Val Tyr Ala His Gln Met     Val     #        380     -      Arg Thr Asp Ser Arg Glu Gln Lys - # Leu Asp Ala Phe Leu Gln Pro     Leu     #    400     -      Ser Lys Pro Leu Ser Ser Gln Pro - # Gln Ala Ile Val Thr Glu Asp     Lys     #   415     -      Thr Asp Ile Ser Ser Gly Arg Ala - # Arg Gln Gln Asp Glu Glu Met     Leu     #                430     -      Glu Leu Pro Ala Pro Ala Glu Val - # Ala Ala Lys Asn Gln Ser Leu     Glu     #            445     -      Gly Asp Thr Thr Lys Gly Thr Ser - # Glu Met Ser Glu Lys Arg Gly     Pro     #        460     -      Thr Ser Ser Asn Pro Arg Lys Arg - # His Arg Glu Asp Ser Asp Val     Glu     #    480     -      Met Val Glu Asp Asp Ser Arg Lys - # Glu Met Thr Ala Ala Cys Thr     Pro     #   495     -      Arg Arg Arg Ile Ile Asn Leu Thr - # Ser Val Leu Ser Leu Gln Glu     Glu     #                510     -      Ile Asn Glu Gln Gly His Glu Val - # Leu Arg Glu Met Leu His Asn     His     #            525     -      Ser Phe Val Gly Cys Val Asn Pro - # Gln Trp Ala Leu Ala Gln His     Gln     #        540     -      Thr Lys Leu Tyr Leu Leu Asn Thr - # Thr Lys Leu Ser Glu Glu Leu     Phe     #    560     -      Tyr Gln Ile Leu Ile Tyr Asp Phe - # Ala Asn Phe Gly Val Leu Arg     Leu     #   575     -      Ser Glu Pro Ala Pro Leu Phe Asp - # Leu Ala Met Leu Ala Leu Asp     Ser     #                590     -      Pro Glu Ser Gly Trp Thr Glu Glu - # Asp Gly Pro Lys Glu Gly Leu     Ala     #            605     -      Glu Tyr Ile Val Glu Phe Leu Lys - # Lys Lys Ala Glu Met Leu Ala     Asp     #        620     -      Tyr Phe Ser Leu Glu Ile Asp Glu - # Glu Gly Asn Leu Ile Gly Leu     Pro     #    640     -      Leu Leu Ile Asp Asn Tyr Val Pro - # Pro Leu Glu Gly Leu Pro Ile     Phe     #   655     -      Ile Leu Arg Leu Ala Thr Glu Val - # Asn Trp Asp Glu Glu Lys Glu     Cys     #                670     -      Phe Glu Ser Leu Ser Lys Glu Cys - # Ala Met Phe Tyr Ser Ile Arg     Lys     #            685     -      Gln Tyr Ile Ser Glu Glu Ser Thr - # Leu Ser Gly Gln Gln Ser Glu     Val     #        700     -      Pro Gly Ser Ile Pro Asn Ser Trp - # Lys Trp Thr Val Glu His Ile     Val     #    720     -      Tyr Lys Ala Leu Arg Ser His Ile - # Leu Pro Pro Lys His Phe Thr     Glu     #   735     -      Asp Gly Asn Ile Leu Gln Leu Ala - # Asn Leu Pro Asp Leu Tyr Lys     Val     #                750     -      Phe Glu Arg Cys                  755     - (2) INFORMATION FOR SEQ ID NO:13:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 770 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:13:(xi) SEQUENCE DESCRIPTION: SEQ     -      Met Ser Leu Arg Ile Lys Ala Leu - # Asp Ala Ser Val Val Asn Lys     Ile     #   15     -      Ala Ala Gly Glu Ile Ile Ile Ser - # Pro Val Asn Ala Leu Lys Glu     Met     #                 30     -      Met Glu Asn Ser Ile Asp Ala Asn - # Ala Thr Met Ile Asp Ile Leu     Val     #             45     -      Lys Glu Gly Gly Ile Lys Val Leu - # Gln Ile Thr Asp Asn Gly Ser     Gly     #         60     -      Ile Asn Lys Ala Asp Leu Pro Ile - # Leu Cys Glu Arg Phe Thr Thr     Ser     #     80     -      Lys Leu Gln Lys Phe Glu Asp Leu - # Ser Gln Ile Gln Thr Tyr Gly     Phe     #   95     -      Arg Gly Glu Ala Leu Ala Ser Ile - # Ser His Val Ala Arg Val Thr     Val     #                110     -      Thr Thr Lys Val Lys Glu Asp Arg - # Cys Ala Trp Arg Val Ser Tyr     Ala     #            125     -      Glu Gly Lys Met Leu Glu Ser Pro - # Lys Pro Val Ala Gly Lys Asp     Gly     #        140     -      Thr Thr Ile Leu Val Glu Asp Leu - # Phe Phe Asn Ile Pro Ser Arg     Leu     #    160     -      Arg Ala Leu Arg Ser His Asn Asp - # Glu Tyr Ser Lys Ile Leu Asp     Val     #   175     -      Val Gly Arg Tyr Ala Ile His Ser - # Lys Asp Ile Gly Phe Ser Cys     Lys     #                190     -      Lys Phe Gly Asp Ser Asn Tyr Ser - # Leu Ser Val Lys Pro Ser Tyr     Thr     #            205     -      Val Gln Asp Arg Ile Arg Thr Val - # Phe Asn Lys Ser Val Ala Ser     Asn     #        220     -      Leu Ile Thr Phe His Ile Ser Lys - # Val Glu Asp Leu Asn Leu Glu     Ser     #    240     -      Val Asp Gly Lys Val Cys Asn Leu - # Asn Phe Ile Ser Lys Lys Ser     Ile     #   255     -      Ser Leu Ile Phe Phe Ile Asn Asn - # Arg Leu Val Thr Cys Asp Leu     Leu     #                270     -      Arg Arg Ala Leu Asn Ser Val Tyr - # Ser Asn Tyr Leu Pro Lys Gly     Phe     #            285     -      Arg Pro Phe Ile Tyr Leu Gly Ile - # Val Ile Asp Pro Ala Ala Val     Asp     #        300     -      Val Asn Val His Pro Thr Lys Arg - # Glu Val Arg Phe Leu Ser Gln     Asp     #    320     -      Glu Ile Ile Glu Lys Ile Ala Asn - # Gln Leu His Ala Glu Leu Ser     Ala     #   335     -      Ile Asp Thr Ser Arg Thr Phe Lys - # Ala Ser Ser Ile Ser Thr Asn     Lys     #                350     -      Pro Glu Ser Leu Ile Pro Phe Asn - # Asp Thr Ile Glu Ser Asp Arg     Asn     #            365     -      Arg Lys Ser Leu Arg Gln Ala Gln - # Val Val Glu Asn Ser Tyr Thr     Thr     #        380     -      Ala Asn Ser Gln Leu Arg Lys Ala - # Lys Arg Gln Glu Asn Lys Leu     Val     #    400     -      Arg Ile Asp Ala Ser Gln Ala Lys - # Ile Thr Ser Phe Leu Ser Ser     Ser     #   415     -      Gln Gln Phe Asn Phe Glu Gly Ser - # Ser Thr Lys Arg Gln Leu Ser     Glu     #                430     -      Pro Lys Val Thr Asn Val Ser His - # Ser Gln Glu Ala Glu Lys Leu     Thr     #            445     -      Leu Asn Glu Ser Glu Gln Pro Arg - # Asp Ala Asn Thr Ile Asn Asp     Asn     #        460     -      Asp Leu Lys Asp Gln Pro Lys Lys - # Lys Gln Lys Gln Leu Gly Asp     Tyr     #    480     -      Lys Val Pro Ser Ile Ala Asp Asp - # Glu Lys Asn Ala Leu Pro Ile     Ser     #   495     -      Lys Asp Gly Tyr Ile Arg Val Pro - # Lys Glu Arg Val Asn Val Asn     Leu     #                510     -      Thr Ser Ile Lys Lys Leu Arg Glu - # Lys Val Asp Asp Ser Ile His     Arg     #            525     -      Glu Leu Thr Asp Ile Phe Ala Asn - # Leu Asn Tyr Val Gly Val Val     Asp     #        540     -      Glu Glu Arg Arg Leu Ala Ala Ile - # Gln His Asp Leu Lys Leu Phe     Leu     #    560     -      Ile Asp Tyr Gly Ser Val Cys Tyr - # Glu Leu Phe Tyr Gln Ile Gly     Leu     #   575     -      Thr Asp Phe Ala Asn Phe Gly Lys - # Ile Asn Leu Gln Ser Thr Asn     Val     #                590     -      Ser Asp Asp Ile Val Leu Tyr Asn - # Leu Leu Ser Glu Phe Asp Glu     Leu     #            605     -      Asn Asp Asp Ala Ser Lys Glu Lys - # Ile Ile Ser Lys Ile Trp Asp     Met     #        620     -      Ser Ser Met Leu Asn Glu Tyr Tyr - # Ser Ile Glu Leu Val Asn Asp     Gly     #    640     -      Leu Asp Asn Asp Leu Lys Ser Val - # Lys Leu Lys Ser Leu Pro Leu     Leu     #   655     -      Leu Lys Gly Tyr Ile Pro Ser Leu - # Val Lys Leu Pro Phe Phe Ile     Tyr     #                670     -      Arg Leu Gly Lys Glu Val Asp Trp - # Glu Asp Glu Gln Glu Cys Leu     Asp     #            685     -      Gly Ile Leu Arg Glu Ile Ala Leu - # Leu Tyr Ile Pro Asp Met Val     Pro     #        700     -      Lys Val Asp Thr Leu Asp Ala Ser - # Leu Ser Glu Asp Glu Lys Ala     Gln     #    720     -      Phe Ile Asn Arg Lys Glu His Ile - # Ser Ser Leu Leu Glu His Val     Leu     #   735     -      Phe Pro Cys Ile Lys Arg Arg Phe - # Leu Ala Pro Arg His Ile Leu     Lys     #                750     -      Asp Val Val Glu Ile Ala Asn Leu - # Pro Asp Leu Tyr Lys Val Phe     Glu     #            765     -      Arg Cys              770     - (2) INFORMATION FOR SEQ ID NO:14:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 64 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:14:(xi) SEQUENCE DESCRIPTION: SEQ     -      Val Asn Arg Ile Ala Ala Gly Glu - # Val Ile Gln Arg Pro Ala Asn     Ala     #   15     -      Ile Lys Glu Met Ile Glu Asn Cys - # Leu Asp Ala Lys Phe Thr Ser     Ile     #                 30     -      Gln Val Ile Val Lys Glu Gly Gly - # Leu Lys Leu Ile Gln Ile Gln     Asp     #             45     -      Asn Gly Thr Gly Ile Arg Lys Glu - # Asp Leu Asp Ile Val Cys Glu     Arg     #         60     - (2) INFORMATION FOR SEQ ID NO:15:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 64 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:15:(xi) SEQUENCE DESCRIPTION: SEQ     -      Val Asn Arg Ile Ala Ala Gly Glu - # Val Ile Gln Arg Pro Ala Asn     Ala     #   15     -      Ile Lys Glu Met Ile Glu Asn Cys - # Leu Asp Ala Lys Ser Thr Ser     Ile     #                 30     -      Gln Val Ile Val Lys Glu Gly Gly - # Leu Lys Leu Ile Gln Ile Gln     Asp     #             45     -      Asn Gly Thr Gly Ile Arg Lys Glu - # Asp Leu Asp Ile Val Cys Glu     Arg     #         60     - (2) INFORMATION FOR SEQ ID NO:16:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 52 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:16:(xi) SEQUENCE DESCRIPTION: SEQ     -      Pro Ala Asn Ala Ile Lys Glu Met - # Ile Glu Asn Cys Leu Asp Ala     Lys     #   15     -      Ser Thr Asn Ile Gln Val Val Val - # Lys Glu Gly Gly Leu Lys Leu     Ile     #                 30     -      Gln Ile Gln Asp Asn Gly Thr Gly - # Ile Arg Lys Glu Asp Leu Asp     Ile     #             45     -      Val Cys Glu Arg              50     - (2) INFORMATION FOR SEQ ID NO:17:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 64 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:17:(xi) SEQUENCE DESCRIPTION: SEQ     -      Val Asn Lys Ile Ala Ala Gly Glu - # Ile Ile Ile Ser Pro Val Asn     Ala     #   15     -      Leu Lys Glu Met Met Glu Asn Ser - # Ile Asp Ala Asn Ala Thr Met     Ile     #                 30     -      Asp Ile Leu Val Lys Glu Gly Gly - # Ile Lys Val Leu Gln Ile Thr     Asp     #             45     -      Asn Gly Ser Gly Ile Asn Lys Ala - # Asp Leu Pro Ile Leu Cys Glu     Arg     #         60     - (2) INFORMATION FOR SEQ ID NO:18:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 64 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:18:(xi) SEQUENCE DESCRIPTION: SEQ     -      Val His Arg Ile Thr Ser Gly Gln - # Val Ile Thr Asp Leu Thr Thr     Ala     #   15     -      Val Lys Glu Leu Val Asp Asn Ser - # Ile Asp Ala Asn Ala Asn Gln     Ile     #                 30     -      Glu Ile Ile Phe Lys Asp Tyr Gly - # Leu Glu Ser Ile Glu Cys Ser     Asp     #             45     -      Asn Gly Asp Gly Ile Asp Pro Ser - # Asn Tyr Glu Phe Leu Ala Leu     Lys     #         60     - (2) INFORMATION FOR SEQ ID NO:19:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 64 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:19:(xi) SEQUENCE DESCRIPTION: SEQ     -      Ala Asn Gln Ile Ala Ala Gly Glu - # Val Val Glu Arg Pro Ala Ser     Val     #   15     -      Val Lys Glu Leu Val Glu Asn Ser - # Leu Asp Ala Gly Ala Thr Arg     Ile     #                 30     -      Asp Ile Asp Ile Glu Arg Gly Gly - # Ala Lys Leu Ile Arg Ile Arg     Asp     #             45     -      Asn Gly Cys Gly Ile Lys Lys Asp - # Glu Leu Ala Leu Ala Leu Ala     Arg     #         60     - (2) INFORMATION FOR SEQ ID NO:20:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 64 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:20:(xi) SEQUENCE DESCRIPTION: SEQ     -      Ala Asn Gln Ile Ala Ala Gly Glu - # Val Val Glu Arg Pro Ala Ser     Val     #   15     -      Val Lys Glu Leu Val Glu Asn Ser - # Leu Asp Ala Gly Ala Thr Arg     Val     #                 30     -      Asp Ile Asp Ile Glu Arg Gly Gly - # Ala Lys Leu Ile Arg Ile Arg     Asp     #             45     -      Asn Gly Cys Gly Ile Lys Lys Glu - # Glu Leu Ala Leu Ala Leu Ala     Arg     #         60     - (2) INFORMATION FOR SEQ ID NO:21:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 64 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:21:(xi) SEQUENCE DESCRIPTION: SEQ     -      Ala Asn Gln Ile Ala Ala Gly Glu - # Val Ile Glu Arg Pro Ala Ser     Val     #   15     -      Cys Lys Glu Leu Val Glu Asn Ala - # Ile Asp Ala Gly Ser Ser Gln     Ile     #                 30     -      Ile Ile Glu Ile Glu Glu Ala Gly - # Leu Lys Lys Val Gln Ile Thr     Asp     #             45     -      Asn Gly His Gly Ile Ala His Asp - # Glu Val Glu Leu Ala Leu Arg     Arg     #         60     - (2) INFORMATION FOR SEQ ID NO:22:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 2687 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: DNA (genomic)     -   (viii) POSITION IN GENOME:               (B) MAP POSITION: 7q     #ID NO:22:(xi) SEQUENCE DESCRIPTION: SEQ     - CCATGGAGCG AGCTGAGAGC TCGAGTACAG AACCTGCTAA GGCCATCAAA CC - #TATTGATC       60     - GGAAGTCAGT CCATCAGATT TGCTCTGGGC AGGTGGTACT GAGTCTAAGC AC - #TGCGGTAA      120     - AGGAGTTAGT AGAAAACAGT CTGGATGCTG GTGCCACTAA TATTGATCTA AA - #GCTTAAGG      180     - ACTATGGAGT GGATCTTATT GAAGTTTCAG ACAATGGATG TGGGGTAGAA GA - #AGAAAACT      240     - TCGAAGGCTT AACTCTGAAA CATCACACAT CTAAGATTCA AGAGTTTGCC GA - #CCTAACTC      300     - AGGTTGAAAC TTTTGGCTTT CGGGGGGAAG CTCTGAGCTC ACTTTGTGCA CT - #GAGCGATG      360     - TCACCATTTC TACCTGCCAC GCATCGGCGA AGGTTGGAAC TCGACTGATG TT - #TGATCACA      420     - ATGGGAAAAT TATCCAGAAA ACCCCCTACC CCCGCCCCAG AGGGACCACA GT - #CAGCGTGC      480     - AGCAGTTATT TTCCACACTA CCTGTGCGCC ATAAGGAATT TCAAAGGAAT AT - #TAAGAAGG      540     - AGTATGCCAA AATGGTCCAG GTCTTACATG CATACTGTAT CATTTCAGCA GG - #CATCCGTG      600     - TAAGTTGCAC CAATCAGCTT GGACAAGGAA AACGACAGCC TGTGGTATGC AC - #AGGTGGAA      660     - GCCCCAGCAT AAAGGAAAAT ATCGGCTCTG TGTTTGGGCA GAAGCAGTTG CA - #AAGCCTCA      720     - TTCCTTTTGT TCAGCTGCCC CCTAGTGACT CCGTGTGTGA AGAGTACGGT TT - #GAGCTGTT      780     - CGGATGCTCT GCATAATCTT TTTTACATCT CAGGTTTCAT TTCACAATGC AC - #GCATGGAG      840     - TTGGAAGGAG TTCAACAGAC AGACAGTTTT TCTTTATCAA CCGGCGGCCT TG - #TGACCCAG      900     - CAAAGGTCTG CAGACTCGTG AATGAGGTCT ACCACATGTA TAATCGACAC CA - #GTATCCAT      960     - TTGTTGTTCT TAACATTTCT GTTGATTCAG AATGCGTTGA TATCAATGTT AC - #TCCAGATA     1020     - AAAGGCAAAT TTTGCTACAA GAGGAAAAGC TTTTGTTGGC AGTTTTAAAG AC - #CTCTTTGA     1080     - TAGGAATGTT TGATAGTGAT GTCAACAAGC TAAATGTCAG TCAGCAGCCA CT - #GCTGGATG     1140     - TTGAAGGTAA CTTAATAAAA ATGCATGCAG CGGATTTGGA AAAGCCCATG GT - #AGAAAAGC     1200     - AGGATCAATC CCCTTCATTA AGGACTGGAG AAGAAAAAAA AGACGTGTCC AT - #TTCCAGAC     1260     - TGCGAGAGGC CTTTTCTCTT CGTCACACAA CAGAGAACAA GCCTCACAGC CC - #AAAGACTC     1320     - CAGAACCAAG AAGGAGCCCT CTAGGACAGA AAAGGGGTAT GCTGTCTTCT AG - #CACTTCAG     1380     - GTGCCATCTC TGACAAAGGC GTCCTGAGAT CTCAGAAAGA GGCAGTGAGT TC - #CAGTCACG     1440     - GACCCAGTGA CCCTACGGAC AGAGCGGAGG TGGAGAAGGA CTCGGGGCAC GG - #CAGCACTT     1500     - CCGTGGATTC TGAGGGGTTC AGCATCCCAG ACACGGGCAG TCACTGCAGC AG - #CGAGTATG     1560     - CGGCCAGCTC CCCAGGGGAC AGGGGCTCGC AGGAACATGT GGACTCTCAG GA - #GAAAGCGC     1620     - CTGAAACTGA CGACTCTTTT TCAGATGTGG ACTGCCATTC AAACCAGGAA GA - #TACCGGAT     1680     - GTAAATTTCG AGTTTTGCCT CAGCCAACTA ATCTCGCAAC CCCAAACACA AA - #GCGTTTTA     1740     - AAAAAGAAGA AATTCTTTCC AGTTCTGACA TTTGTCAAAA GTTAGTAAAT AC - #TCAGGACA     1800     - TGTCAGCCTC TCAGGTTGAT TGAGCTGTGA AAATTAATAA GAAAGTTGTG CC - #CCTGGACT     1860     - TTTCTATGAG TTCTTTAGCT AAACGAATAA AGCAGTTACA TCATGAAGCA CA - #GCAAAGTG     1920     - AAGGGGAACA GAATTACAGG AAGTTTAGGG CAAAGATTTG TCCTGGAGAA AA - #TCAAGCAG     1980     - CCGAAGATGA ACTAAGAAAA GAGATAAGTA AAACGATGTT TGCAGAAATG GA - #AATCATTG     2040     - GTCAGTTTAA CCTGGGATTT ATAATAACCA AACTGAATGA GGATATCTTC AT - #AGTGGACC     2100     - AGCATGCCAC GGACGAGAAG TATAACTTCG AGATGCTGCA GCAGCACACC GT - #GCTCCAGG     2160     - GGCAGAGGCT CATAGCACCT CAGACTCTCA ACTTAACTGC TGTTAATGAA GC - #TGTTCTGA     2220     - TAGAAAATCT GGAAATATTT AGAAAGAATG GCTTTGATTT TGTTATCGAT GA - #AAATGCTC     2280     - CAGTCACTGA AAGGGCTAAA CTGATTTCCT TGCCAACTAG TAAAAACTGG AC - #CTTCGGAC     2340     - CCCAGGACGT CGATGAACTG ATCTTCATGC TGAGCGACAG CCCTGGGGTC AT - #GTGCCGCC     2400     - CTTCCCGAGT CAAGCAGATG TTTGCCTCCA GAGCCTGCCG GAAGTCGGTG AT - #GATTGGGA     2460     - CTGCTCTCAA CACAAGCGAA TGAAGAAACT GATCACCCAC ATGGGGGAGA TG - #GGCCACCC     2520     - CTGGAACTGT CCCCATGGAA GGCCACCATG AGACACATCG CCAACCTGGG TG - #TCATTTCT     2580     - CAGAACTGAC CGTAGTCACT GTATGGAATA ATTGGTTTTA TCGCAGATTT TT - #ATGTTTTG     2640     #              2687CTAA CCTTTTTTGT TTTAAAATGA AACCTGC     - (2) INFORMATION FOR SEQ ID NO:23:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 862 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:23:(xi) SEQUENCE DESCRIPTION: SEQ     -      Met Glu Arg Ala Glu Ser Ser Ser - # Thr Glu Pro Ala Lys Ala Ile     Lys     #   15     -      Pro Ile Asp Arg Lys Ser Val His - # Gln Ile Cys Ser Gly Gln Val     Val     #                 30     -      Leu Ser Leu Ser Thr Ala Val Lys - # Glu Leu Val Glu Asn Ser Leu     Asp     #             45     -      Ala Gly Ala Thr Asn Ile Asp Leu - # Lys Leu Lys Asp Tyr Gly Val     Asp     #         60     -      Leu Ile Glu Val Ser Asp Asn Gly - # Cys Gly Val Glu Glu Glu Asn     Phe     #     80     -      Glu Gly Leu Thr Leu Lys His His - # Thr Ser Lys Ile Gln Glu Phe     Ala     #   95     -      Asp Leu Thr Gln Val Glu Thr Phe - # Gly Phe Arg Gly Glu Ala Leu     Ser     #                110     -      Ser Leu Cys Ala Leu Ser Asp Val - # Thr Ile Ser Thr Cys His Ala     Ser     #            125     -      Ala Lys Val Gly Thr Arg Leu Met - # Phe Asp His Asn Gly Lys Ile     Ile     #        140     -      Gln Lys Thr Pro Tyr Pro Arg Pro - # Arg Gly Thr Thr Val Ser Val     Gln     #    160     -      Gln Leu Phe Ser Thr Leu Pro Val - # Arg His Lys Glu Phe Gln Arg     Asn     #   175     -      Ile Lys Lys Glu Tyr Ala Lys Met - # Val Gln Val Leu His Ala Tyr     Cys     #                190     -      Ile Ile Ser Ala Gly Ile Arg Val - # Ser Cys Thr Asn Gln Leu Gly     Gln     #            205     -      Gly Lys Arg Gln Pro Val Val Cys - # Ile Gly Gly Ser Pro Ser Ile     Lys     #        220     -      Glu Asn Ile Gly Ser Val Phe Gly - # Gln Lys Gln Leu Gln Ser Leu     Ile     #    240     -      Pro Phe Val Gln Leu Pro Pro Ser - # Asp Ser Val Cys Glu Glu Tyr     Gly     #   255     -      Leu Ser Cys Ser Asp Ala Leu His - # Asn Leu Phe Tyr Ile Ser Gly     Phe     #                270     -      Ile Ser Gln Cys Thr His Gly Val - # Gly Arg Ser Ser Thr Asp Arg     Gln     #            285     -      Phe Phe Phe Ile Asn Arg Arg Pro - # Cys Asp Pro Ala Lys Val Cys     Arg     #        300     -      Leu Val Asn Glu Val Tyr His Met - # Tyr Asn Arg His Gln Tyr Pro     Phe     #    320     -      Val Val Leu Asn Ile Ser Val Asp - # Ser Glu Cys Val Asp Ile Asn     Val     #   335     -      Thr Pro Asp Lys Arg Gln Ile Leu - # Leu Gln Glu Glu Lys Leu Leu     Leu     #                350     -      Ala Val Leu Lys Thr Ser Leu Ile - # Gly Met Phe Asp Ser Asp Val     Asn     #            365     -      Lys Leu Asn Val Ser Gln Gln Pro - # Leu Leu Asp Val Glu Gly Asn     Leu     #        380     -      Ile Lys Met His Ala Ala Asp Leu - # Glu Lys Pro Met Val Glu His     Gln     #    400     -      Asp Gln Ser Pro Ser Leu Arg Ile - # Gly Glu Glu Lys Lys Asp Val     Ser     #   415     -      Ile Ser Arg Leu Arg Glu Ala Phe - # Ser Leu Arg His Thr Thr Glu     Asn     #                430     -      Lys Pro His Ser Pro Lys Thr Pro - # Glu Pro Arg Arg Ser Pro Leu     Gly     #            445     -      Gln Lys Arg Gly Met Leu Ser Ser - # Ser Thr Ser Gly Ala Ile Ser     Asp     #        460     -      Lys Gly Val Leu Arg Ser Gln Lys - # Glu Ala Val Ser Ser Ser His     Gly     #    480     -      Pro Ser Asp Pro Thr Asp Arg Ala - # Glu Val Glu Lys Asp Ser Gly     His     #   495     -      Gly Ser Thr Ser Val Asp Ser Glu - # Gly Phe Ser Ile Pro Asp Thr     Gly     #                510     -      Ser His Cys Ser Ser Glu Tyr Ala - # Ala Ser Ser Pro Gly Asp Arg     Gly     #            525     -      Ser Gln Glu His Val Asp Ser Gln - # Glu Lys Ala Pro Glu Thr Asp     Asp     #        540     -      Ser Phe Ser Asp Val Asp Cys His - # Ser Asn Gln Glu Asp Thr Gly     Cys     #    560     -      Lys Phe Arg Val Leu Pro Gln Pro - # Ile Asn Leu Ala Thr Pro Asn     Thr     #   575     -      Lys Arg Phe Lys Lys Glu Glu Ile - # Leu Ser Ser Ser Asp Ile Cys     Gln     #                590     -      Lys Leu Val Asn Thr Gln Asp Met - # Ser Ala Ser Gln Val Asp Val     Ala     #            605     -      Val Lys Ile Asn Lys Lys Val Val - # Pro Leu Asp Phe Ser Met Ser     Ser     #        620     -      Leu Ala Lys Arg Ile Lys Gln Leu - # His His Glu Ala Gln Gln Ser     Glu     #    640     -      Gly Glu Gln Asn Tyr Arg Lys Phe - # Arg Ala Lys Ile Cys Pro Gly     Glu     #   655     -      Asn Gln Ala Ala Glu Asp Glu Leu - # Arg Lys Glu Ile Ser Lys Thr     Met     #                670     -      Phe Ala Glu Met Glu Ile Ile Gly - # Gln Phe Asn Leu Gly Phe Ile     Ile     #            685     -      Thr Lys Leu Asn Glu Asp Ile Phe - # Ile Val Asp Gln His Ala Thr     Asp     #        700     -      Glu Lys Tyr Asn Phe Glu Met Leu - # Gln Gln His Thr Val Leu Gln     Gly     #    720     -      Gln Arg Leu Ile Ala Pro Gln Thr - # Leu Asn Leu Thr Ala Val Asn     Glu     #   735     -      Ala Val Leu Ile Glu Asn Leu Glu - # Ile Phe Arg Lys Asn Gly Phe     Asp     #                750     -      Phe Val Ile Asp Glu Asn Ala Pro - # Val Thr Glu Arg Ala Lys Leu     Ile     #            765     -      Ser Leu Pro Thr Ser Lys Asn Trp - # Thr Phe Gly Pro Gln Asp Val     Asp     #        780     -      Glu Leu Ile Phe Met Leu Ser Asp - # Ser Pro Gly Val Met Cys Arg     Pro     #    800     -      Ser Arg Val Lys Gln Met Phe Ala - # Ser Arg Ala Cys Arg Lys Ser     Val     #   815     -      Met Ile Gly Thr Ala Leu Asn Thr - # Ser Glu Met Lys Lys Leu Ile     Thr     #                830     -      His Met Gly Glu Met Gly His Pro - # Trp Asn Cys Pro His Gly Arg     Pro     #            845     -      Thr Met Arg His Ile Ala Asn Leu - # Gly Val Ile Ser Gln Asn     #        860     - (2) INFORMATION FOR SEQ ID NO:24:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 903 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:24:(xi) SEQUENCE DESCRIPTION: SEQ     -      Met Phe His His Ile Glu Asn Leu - # Leu Ile Glu Thr Glu Lys Arg     Cys     #   15     -      Lys Gln Lys Glu Gln Arg Tyr Ile - # Pro Val Lys Tyr Leu Phe Ser     Met     #                 30     -      Thr Gln Ile His Gln Ile Asn Asp - # Ile Asp Val His Arg Ile Thr     Ser     #             45     -      Gly Gln Val Ile Thr Asp Leu Thr - # Thr Ala Val Lys Glu Leu Val     Asp     #         60     -      Asn Ser Ile Asp Ala Asn Ala Asn - # Gln Ile Glu Ile Ile Phe Lys     Asp     #     80     -      Tyr Gly Leu Glu Ser Ile Glu Cys - # Ser Asp Asn Gly Asp Gly Ile     Asp     #   95     -      Pro Ser Asn Tyr Glu Phe Leu Ala - # Leu Lys His Tyr Thr Ser Lys     Ile     #                110     -      Ala Lys Phe Gln Asp Val Ala Lys - # Val Gln Thr Leu Gly Phe Arg     Gly     #            125     -      Glu Ala Leu Ser Ser Leu Cys Gly - # Ile Ala Lys Leu Ser Val Ile     Thr     #        140     -      Thr Thr Ser Pro Pro Lys Ala Asp - # Lys Leu Glu Tyr Asp Met Val     Gly     #    160     -      His Ile Thr Ser Lys Thr Thr Ser - # Arg Asn Lys Gly Thr Thr Val     Leu     #   175     -      Val Ser Gln Leu Phe His Asn Leu - # Pro Val Arg Gln Lys Glu Phe     Ser     #                190     -      Lys Thr Phe Lys Arg Gln Phe Thr - # Lys Cys Leu Thr Val Ile Gln     Gly     #            205     -      Tyr Ala Ile Ile Asn Ala Ala Ile - # Lys Phe Ser Val Trp Asn Ile     Thr     #        220     -      Pro Lys Gly Lys Lys Asn Leu Ile - # Leu Ser Thr Met Arg Asn Ser     Ser     #    240     -      Met Arg Lys Asn Ile Ser Ser Val - # Phe Gly Ala Gly Gly Met Phe     Gly     #   255     -      Leu Glu Glu Val Asp Leu Val Leu - # Asp Leu Asn Pro Phe Lys Asn     Arg     #                270     -      Met Leu Gly Lys Tyr Thr Asp Asp - # Pro Asp Phe Leu Asp Leu Asp     Tyr     #            285     -      Lys Ile Arg Val Lys Gly Tyr Ile - # Ser Gln Asn Ser Phe Gly Cys     Gly     #        300     -      Arg Asn Ser Lys Asp Arg Gln Phe - # Ile Tyr Val Asn Lys Arg Pro     Val     #    320     -      Glu Tyr Ser Thr Leu Leu Lys Cys - # Cys Asn Glu Val Tyr Lys Thr     Phe     #   335     -      Asn Asn Val Gln Phe Pro Ala Val - # Phe Leu Asn Leu Glu Leu Pro     Met     #                350     -      Ser Leu Ile Asp Val Asn Val Thr - # Pro Asp Lys Arg Val Ile Leu     Leu     #            365     -      His Asn Glu Arg Ala Val Ile Asp - # Ile Phe Lys Thr Thr Leu Ser     Asp     #        380     -      Tyr Tyr Asn Arg Gln Glu Leu Ala - # Leu Pro Lys Arg Met Cys Ser     Gln     #    400     -      Ser Glu Gln Gln Ala Gln Lys Arg - # Leu Lys Thr Glu Val Phe Asp     Asp     #   415     -      Arg Ser Thr Thr His Glu Ser Asp - # Asn Glu Asn Tyr His Thr Ala     Arg     #                430     -      Ser Glu Ser Asn Gln Ser Asn His - # Ala His Phe Asn Ser Thr Thr     Gly     #            445     -      Val Ile Asp Lys Ser Asn Gly Thr - # Glu Leu Thr Ser Val Met Asp     Gly     #        460     -      Asn Tyr Thr Asn Val Thr Asp Val - # Ile Gly Ser Glu Cys Glu Val     Ser     #    480     -      Val Asp Ser Ser Val Val Leu Asp - # Glu Gly Asn Ser Ser Thr Pro     Thr     #   495     -      Lys Lys Leu Pro Ser Ile Lys Thr - # Asp Ser Gln Asn Leu Ser Asp     Leu     #                510     -      Asn Leu Asn Asn Phe Ser Asn Pro - # Glu Phe Gln Asn Ile Thr Ser     Pro     #            525     -      Asp Lys Ala Arg Ser Leu Glu Lys - # Val Val Glu Glu Pro Val Tyr     Phe     #        540     -      Asp Ile Asp Gly Glu Lys Phe Gln - # Glu Lys Ala Val Leu Ser Gln     Ala     #    560     -      Asp Gly Leu Val Phe Val Asp Asn - # Glu Cys His Glu His Thr Asn     Asp     #   575     -      Cys Cys His Gln Glu Arg Arg Gly - # Ser Thr Asp Ile Glu Gln Asp     Asp     #                590     -      Glu Ala Asp Ser Ile Tyr Ala Glu - # Ile Glu Pro Val Glu Ile Asn     Val     #            605     -      Arg Thr Pro Leu Lys Asn Ser Arg - # Lys Ser Ile Ser Lys Asp Asn     Tyr     #        620     -      Arg Ser Leu Ser Asp Gly Leu Thr - # His Arg Lys Phe Glu Asp Glu     Ile     #    640     -      Leu Glu Tyr Asn Leu Ser Thr Lys - # Asn Phe Lys Glu Ile Ser Lys     Asn     #   655     -      Gly Lys Gln Met Ser Ser Ile Ile - # Ser Lys Arg Lys Ser Glu Ala     Gln     #                670     -      Glu Asn Ile Ile Lys Asn Lys Asp - # Glu Leu Glu Asp Phe Glu Gln     Gly     #            685     -      Glu Lys Tyr Leu Thr Leu Thr Val - # Ser Lys Asn Asp Phe Lys Lys     Met     #        700     -      Glu Val Val Gly Gln Phe Asn Leu - # Gly Phe Ile Ile Val Thr Arg     Lys     #    720     -      Val Asp Asn Lys Ser Lys Leu Phe - # Ile Val Asp Gln His Ala Ser     Asp     #   735     -      Glu Lys Tyr Asn Phe Glu Thr Leu - # Gln Ala Val Thr Val Phe Lys     Ser     #                750     -      Gln Lys Leu Ile Ile Pro Gln Pro - # Val Glu Leu Ser Val Ile Asp     Glu     #            765     -      Leu Val Val Leu Asp Asn Leu Pro - # Val Phe Glu Lys Asn Gly Phe     Lys     #        780     -      Leu Lys Ile Asp Glu Glu Glu Glu - # Phe Gly Ser Arg Val Lys Leu     Leu     #    800     -      Ser Leu Pro Thr Ser Lys Gln Thr - # Leu Phe Asp Leu Gly Asp Phe     Asn     #   815     -      Glu Leu Ile His Leu Ile Lys Glu - # Asp Gly Gly Leu Arg Arg Asp     Asn     #                830     -      Ile Arg Cys Ser Lys Ile Arg Ser - # Met Phe Ala Met Arg Ala Cys     Arg     #            845     -      Ser Ser Ile Met Ile Gly Lys Pro - # Leu Asn Lys Lys Thr Met Thr     Arg     #        860     -      Val Val His Asn Leu Ser Glu Leu - # Asp Lys Pro Trp Asn Cys Pro     His     #    880     -      Gly Arg Pro Thr Met Arg His Leu - # Met Glu Ile Arg Asp Trp Ser     Ser     #   895     -      Phe Ser Lys Asp Tyr Glu Ile                      900     - (2) INFORMATION FOR SEQ ID NO:25:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 2577 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     -   (viii) POSITION IN GENOME:               (B) MAP POSITION: Chrom - #osome 9 Band E     #ID NO:25:(xi) SEQUENCE DESCRIPTION: SEQ     - TTCCGGCCAA TGCTATCAAA GAGATGATAG AAAACTGTTT AGATGCAAAA TC - #TACAAATA       60     - TTCAAGTGGT TGTTAAGGAA GGTGGCCTGA AGCTAATTCA GATCCAAGAC AA - #TGGCACTG      120     - GAATCAGGAA GGAAGATCTG GATATTGTGT GTGAGAGGTT CACTACGAGT AA - #ACTGCAGA      180     - CTTTTGAGGA TTTAGCCAGT ATTTCTACCT ATGGCTTTCG TGGTGAGCAT TT - #GGCAAGCA      240     - TAAGTCATGT GGCCCATGTC ACTATTACAA CCAAAACAGC TGATGGGAAA TG - #TGCGTACA      300     - GAGCAAGTTA CTCAGATGGA AAGCTGCAAG CCCCTCCTAA ACCCTGTGCA GG - #CAACCAGG      360     - GCACCCTGAT CACGGTGGAA GACCTTTTTT ACAACATAAT CACAAGGAGG AA - #AGCTTTAA      420     - AAAATCCAAG TGAAGAGTAC GGAAAAATTT TGGAAGTTGT TGGCAGGTAT TC - #AATACACA      480     - ATTCAGGCAT TAGTATCTCA GTTAAAAAAC AAGGTGAGAC AGTATCTGAT GT - #CAGAACAC      540     - TGCCCAATGC CACAACCGTG GACAACATTC GCTCCATCTT TGGAAATGCG GT - #TAGTCGAG      600     - AACTGATAGA AGTTGGGTGT GAGGATAAAA CCCTAGCTTT CAAAATGAAT GG - #CTATATAT      660     - CGAATGCAAA GTATTCAGTG AAGAAGTGCA TTTTCCTACT CTTCATCAAC CA - #CCGTCTGG      720     - TAGAATCAGC TGCCTTGAGA AAAGCCATTG AAACTGTATA TGCAGCATAC TT - #GCCAAAAA      780     - CACACACCCA TTCCTGTACC TCAGTTTGAA ATCAGCCCTC AGAACGTGAC GT - #CAATGTAC      840     - ACCCCACCAA GACAGAAGTT CATTTTCTGC ACGAGGAGAG CATTCTGCAG CG - #TGTGCAGC      900     - AGCACATTGA GAGCAAGCTG CTGGGCTCCA ATTCCTCCAG GATGTATTTC AC - #CCAGACCT      960     - TGCTTCCAGG ACTTGCTGGG CCTCTGGGGA GGCAGCTAGA CCCACGACAG GG - #GTGGCTTC     1020     - CTCATCCACT AGTGGAAGTG GCGACAAGGT CTACGCTTAC CAGATGTCGC GT - #ACGGACTC     1080     - CCGGGATCAG AAGCTTGACG CCTTTCTGCA GCCTGTAAGC AGCCTTGTGC CC - #AGCCAGCC     1140     - CCAGGACCCT CGCCCTGTCC GAGGGGCCAG GACAGAGGGC TCTCCTGAAA GG - #GCCACGCG     1200     - GGAGGATGAG GAGATGCTTG CTCTCCCAGC CCCCGCTGAA GCAGCTGCTG AG - #AGTGAGAA     1260     - CTTGGAGAGG GAATCACTAA TGGAGACTTC AGACGCAGCC CAGAAAGCGG CA - #CCCACTTC     1320     - CAGTCCAGGA AGCTCCAGAA AGAGTCATCG GGAGGACTCT GATGTGGAAA TG - #GTGGAAAA     1380     - TGCTTCCGGG AAGGAAATGA CAGCTGCTTG CTACCCCAGG AGGAGGATCA TT - #AACCTCAC     1440     - CAGCGTCTTG AGTCTCCAGG AAGAGATTAG TGAGCGGTGC CATGAGACTC TC - #CGGGAGAT     1500     - ACTCCGTAAC CATTCCTTTG TGGGCTGTGT GAATCCTCAG TGGGCCTTGG CA - #CAGCACCA     1560     - GACCAAGCTA TACCTCCTCA ACACTACCAA GCTCAGTGAA GAGCTGTTCT AC - #CAGATACT     1620     - CATTTATGAT TTTGCCAACT TTGGTGTTCT GAGGTTATCG GAACCAGCGC CA - #CTCTTCGA     1680     - CCTGGCCATG CTGGCTTAGA CAGTCCTGAA AGTGGCTGGA CAGAGGACGA CG - #GCCCGAAG     1740     - AAGGGCTTGC AGAGTACATT GTCGAGTTTC TGAAGAGAAG CGAGATGCTT GC - #AGACTATT     1800     - CTCTGTGAGA TCGATGAGAA GGGAACCTGA TTGATTACTC TTCTGATGAC AG - #CTATGTGC     1860     - CACCTTTGGA GGGACTGCCT ATCTTCATTC TTCGACTGGC CACTGAGGTG AA - #TTGGGTGA     1920     - AGAAAAGGAG TGTTTTGAAA GTCTCAGTAA AGAATGTGCT ATGTTTTACT CC - #ATTCGGAA     1980     - GCAGTATATA CTGGAGGAGT CGACCCTCTC AGGCCAGCAG AGTGACATGC CT - #GGCTCCAC     2040     - GTCAAAGCCC TGGAAGTGGA CTGTGGAGCA CATTATCTAT AAAGCCTTCC GC - #TCACACCT     2100     - CCTACCTCCG AAGCATTTCA CAGAAGATGG CAATGTCCTG CAGCTTGCCA AC - #CTGCCAGA     2160     - TCTATACAAA GTCTTTGAGC GGTGTTAAAT ACAATCATAG CCACCGTAGA GA - #CTGCATGA     2220     - CCATCCAAGG CGAAGTGTAT GGTACTAATC TGGAAGCCAC AGAATAGGAC AC - #TTGGTTTC     2280     - AGCTCCAGGG TTTTCAGTGC TCACTATTCT TGTTCTGTAT CCCAGTATTG GT - #GCTGCAAC     2340     - TTAATGTACT TCACCTGTGG ATTGGCTGCA AATAAACTCA CGTGTATTGG AA - #AAAAGGAA     2400     - TTCCTGCAGC CCGGGGGATC CACTAGTTCT AGAGCGGCCG CCACCGGTGG AG - #CTCCAGCT     2460     - TTTGTTCCCT TTAGTGAGGG TTAATTTCGA GCTTGGCGTA ATCATGGTCA TA - #GCTGTTTC     2520     - CTGTGTGAAA TTGTTATCCG CTCACAATTC CACACAACAT ACGAGCCGGA AG - #CATAA     2577     - (2) INFORMATION FOR SEQ ID NO:26:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 728 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:26:(xi) SEQUENCE DESCRIPTION: SEQ     -      Pro Ala Asn Ala Ile Lys Glu Met - # Ile Glu Asn Cys Leu Asp Ala     Lys     #   15     -      Ser Thr Asn Ile Gln Val Val Val - # Lys Glu Gly Gly Leu Lys Leu     Ile     #                 30     -      Gln Ile Gln Asp Asn Gly Thr Gly - # Ile Arg Lys Glu Asp Leu Asp     Ile     #             45     -      Val Cys Glu Arg Phe Thr Thr Ser - # Lys Leu Gln Thr Phe Glu Asp     Leu     #         60     -      Ala Ser Ile Ser Thr Tyr Gly Phe - # Arg Gly Glu His Leu Ala Ser     Ile     #     80     -      Ser His Val Ala His Val Thr Ile - # Thr Thr Lys Thr Ala Asp Gly     Lys     #   95     -      Cys Ala Tyr Arg Ala Ser Tyr Ser - # Asp Gly Lys Leu Gln Ala Pro     Pro     #                110     -      Lys Pro Cys Ala Gly Asn Gln Gly - # Thr Leu Ile Thr Val Glu Asp     Leu     #            125     -      Phe Tyr Asn Ile Ile Thr Arg Arg - # Lys Ala Leu Lys Asn Pro Ser     Glu     #        140     -      Glu Tyr Gly Lys Ile Leu Glu Val - # Val Gly Arg Tyr Ser Ile His     Asn     #    160     -      Ser Gly Ile Ser Ile Ser Val Lys - # Lys Gln Gly Glu Thr Val Ser     Asp     #   175     -      Val Arg Thr Leu Pro Asn Ala Thr - # Thr Val Asp Asn Ile Arg Ser     Ile     #                190     -      Phe Gly Asn Ala Val Ser Arg Glu - # Leu Ile Glu Val Gly Cys Glu     Asp     #            205     -      Lys Thr Leu Ala Phe Lys Met Asn - # Gly Tyr Ile Ser Asn Ala Lys     Tyr     #        220     -      Ser Val Lys Lys Cys Ile Phe Leu - # Leu Phe Ile Asn His Arg Leu     Val     #    240     -      Glu Ser Ala Ala Leu Arg Lys Ala - # Ile Glu Thr Val Tyr Ala Ala     Tyr     #   255     -      Leu Pro Lys Thr His Thr His Ser - # Cys Thr Ser Val Glx Asn Gln     Pro     #                270     -      Ser Glu Arg Asp Val Asn Val His - # Pro Thr Lys Thr Glu Val His     Phe     #            285     -      Leu His Glu Glu Ser Ile Leu Gln - # Arg Val Gln Gln His Ile Glu     Ser     #        300     -      Lys Leu Leu Gly Ser Asn Ser Ser - # Arg Met Val Phe His Pro Lys     Leu     #    320     -      Ala Ser Arg Thr Cys Trp Ala Ser - # Gly Glu Ala Ala Arg Pro Thr     Thr     #   335     -      Gly Val Ala Ser Ser Ser Thr Ser - # Gly Ser Gly Asp Lys Val Tyr     Ala     #                350     -      Tyr Gln Met Ser Arg Thr Asp Ser - # Arg Asp Gln Lys Leu Asp Ala     Phe     #            365     -      Leu Gln Pro Val Ser Ser Leu Val - # Pro Ser Gln Pro Gln Asp Pro     Arg     #        380     -      Pro Val Arg Gly Ala Arg Thr Glu - # Gly Ser Pro Glu Arg Ala Thr     Arg     #    400     -      Glu Asp Glu Glu Met Leu Ala Leu - # Pro Ala Pro Ala Glu Ala Ala     Ala     #   415     -      Glu Ser Glu Asn Leu Glu Arg Glu - # Ser Leu Met Glu Thr Ser Asp     Ala     #                430     -      Ala Gln Lys Ala Ala Pro Thr Ser - # Ser Pro Gly Ser Ser Arg Lys     Ser     #            445     -      His Arg Glu Asp Ser Asp Val Glu - # Met Val Glu Asn Ala Ser Gly     Lys     #        460     -      Glu Met Thr Ala Ala Cys Tyr Pro - # Arg Arg Arg Ile Ile Asn Leu     Thr     #    480     -      Ser Val Leu Ser Leu Gln Glu Glu - # Ile Ser Glu Arg Cys His Glu     Thr     #   495     -      Leu Arg Glu Ile Leu Arg Asn His - # Ser Phe Val Gly Cys Val Asn     Pro     #                510     -      Gln Trp Ala Leu Ala Gln His Gln - # Thr Lys Leu Tyr Leu Leu Asn     Thr     #            525     -      Thr Lys Leu Ser Glu Glu Leu Phe - # Tyr Gln Ile Leu Ile Tyr Asp     Phe     #        540     -      Ala Asn Phe Gly Val Leu Arg Leu - # Ser Glu Pro Ala Pro Leu Phe     Asp     #    560     -      Leu Ala Met Leu Ala Glx Thr Val - # Leu Lys Val Ala Gly Gln Arg     Thr     #   575     -      Thr Ala Arg Arg Arg Ala Cys Arg - # Val His Cys Arg Val Ser Glu     Glu     #                590     -      Lys Arg Asp Ala Cys Arg Leu Phe - # Ser Val Arg Ser Met Arg Arg     Glu     #            605     -      Pro Asp Glx Leu Leu Phe Glx Glx - # Gln Leu Cys Ala Thr Phe Gly     Gly     #        620     -      Thr Ala Tyr Leu His Ser Ser Thr - # Gly His Glx Gly Glu Leu Gly     Glu     #    640     -      Glu Lys Glu Cys Phe Glu Ser Leu - # Ser Lys Glu Cys Ala Met Phe     Tyr     #   655     -      Ser Ile Arg Lys Gln Tyr Ile Leu - # Glu Glu Ser Thr Leu Ser Gly     Gln     #                670     -      Gln Ser Asp Met Pro Gly Ser Thr - # Ser Lys Pro Trp Lys Trp Thr     Val     #            685     -      Glu His Ile Ile Tyr Lys Ala Phe - # Arg Ser His Leu Leu Pro Pro     Lys     #        700     -      His Phe Thr Glu Asp Gly Asn Val - # Leu Gln Leu Ala Asn Leu Pro     Asp     #    720     -      Leu Tyr Lys Val Phe Glu Arg Cys     #   725     - (2) INFORMATION FOR SEQ ID NO:27:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 756 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:27:(xi) SEQUENCE DESCRIPTION: SEQ     -      Met Ser Phe Val Ala Gly Val Ile - # Arg Arg Leu Asp Glu Thr Val     Val     #   15     -      Asn Arg Ile Ala Ala Gly Glu Val - # Ile Gln Arg Pro Ala Asn Ala     Ile     #                 30     -      Lys Glu Met Ile Glu Asn Cys Leu - # Asp Ala Lys Ser Thr Ser Ile     Gln     #             45     -      Val Ile Val Lys Glu Gly Gly Leu - # Lys Leu Ile Gln Ile Gln Asp     Asn     #         60     -      Gly Thr Gly Ile Arg Lys Glu Asp - # Leu Asp Ile Val Cys Glu Arg     Phe     #     80     -      Thr Thr Ser Lys Leu Gln Ser Phe - # Glu Asp Leu Ala Ser Ile Ser     Thr     #   95     -      Tyr Gly Phe Arg Gly Glu Ala Leu - # Ala Ser Ile Ser His Val Ala     His     #                110     -      Val Thr Ile Thr Thr Lys Thr Ala - # Asp Gly Lys Cys Ala Tyr Arg     Ala     #            125     -      Ser Tyr Ser Asp Gly Lys Leu Lys - # Ala Pro Pro Lys Pro Cys Ala     Gly     #        140     -      Asn Gln Gly Thr Gln Ile Thr Val - # Glu Asp Leu Phe Tyr Asn Ile     Ala     #    160     -      Thr Arg Arg Lys Ala Leu Lys Asn - # Pro Ser Glu Glu Tyr Gly Lys     Ile     #   175     -      Leu Glu Val Val Gly Arg Tyr Ser - # Val His Asn Ala Gly Ile Ser     Phe     #                190     -      Ser Val Lys Lys Gln Gly Glu Thr - # Val Ala Asp Val Arg Thr Leu     Pro     #            205     -      Asn Ala Ser Thr Val Asp Asn Ile - # Arg Ser Ile Phe Gly Asn Ala     Val     #        220     -      Ser Arg Glu Leu Ile Glu Ile Gly - # Cys Glu Asp Lys Thr Leu Ala     Phe     #    240     -      Lys Met Asn Gly Tyr Ile Ser Asn - # Ala Asn Tyr Ser Val Lys Lys     Cys     #   255     -      Ile Phe Leu Leu Phe Ile Asn His - # Arg Leu Val Glu Ser Thr Ser     Leu     #                270     -      Arg Lys Ala Ile Glu Thr Val Tyr - # Ala Ala Tyr Leu Pro Lys Asn     Thr     #            285     -      His Pro Phe Leu Tyr Leu Ser Leu - # Glu Ile Ser Pro Gln Asn Val     Asp     #        300     -      Val Asn Val His Pro Thr Lys His - # Glu Val His Phe Leu His Glu     Glu     #    320     -      Ser Ile Leu Glu Arg Val Gln Gln - # His Ile Glu Ser Lys Leu Leu     Gly     #   335     -      Ser Asn Ser Ser Arg Met Tyr Phe - # Thr Gln Thr Leu Leu Pro Gly     Leu     #                350     -      Ala Gly Pro Ser Gly Glu Met Val - # Lys Ser Thr Thr Ser Leu Thr     Ser     #            365     -      Ser Ser Thr Ser Gly Ser Ser Asp - # Lys Val Tyr Ala His Gln Met     Val     #        380     -      Arg Thr Asp Ser Arg Glu Gln Lys - # Leu Asp Ala Phe Leu Gln Pro     Leu     #    400     -      Ser Lys Pro Leu Ser Ser Gln Pro - # Gln Ala Ile Val Thr Glu Asp     Lys     #   415     -      Thr Asp Ile Ser Ser Gly Arg Ala - # Arg Gln Gln Asp Glu Glu Met     Leu     #                430     -      Glu Leu Pro Ala Pro Ala Glu Val - # Ala Ala Lys Asn Gln Ser Leu     Glu     #            445     -      Gly Asp Thr Thr Lys Gly Thr Ser - # Glu Met Ser Glu Lys Arg Gly     Pro     #        460     -      Thr Ser Ser Asn Pro Arg Lys Arg - # His Arg Glu Asp Ser Asp Val     Glu     #    480     -      Met Val Glu Asp Asp Ser Arg Lys - # Glu Met Thr Ala Ala Cys Thr     Pro     #   495     -      Arg Arg Arg Ile Ile Asn Leu Thr - # Ser Val Leu Ser Leu Gln Glu     Glu     #                510     -      Ile Asn Glu Gln Gly His Glu Val - # Leu Arg Glu Met Leu His Asn     His     #            525     -      Ser Phe Val Gly Cys Val Asn Pro - # Gln Trp Ala Leu Ala Gln His     Gln     #        540     -      Thr Lys Leu Tyr Leu Leu Asn Thr - # Thr Lys Leu Ser Glu Glu Leu     Phe     #    560     -      Tyr Gln Ile Leu Ile Tyr Asp Phe - # Ala Asn Phe Gly Val Leu Arg     Leu     #   575     -      Ser Glu Pro Ala Pro Leu Phe Asp - # Leu Ala Met Leu Ala Leu Asp     Ser     #                590     -      Pro Glu Ser Gly Trp Thr Glu Glu - # Asp Gly Pro Lys Glu Gly Leu     Ala     #            605     -      Glu Tyr Ile Val Glu Phe Leu Lys - # Lys Lys Ala Glu Met Leu Ala     Asp     #        620     -      Tyr Phe Ser Leu Glu Ile Asp Glu - # Glu Gly Asn Leu Ile Gly Leu     Pro     #    640     -      Leu Leu Ile Asp Asn Tyr Val Pro - # Pro Leu Glu Gly Leu Pro Ile     Phe     #   655     -      Ile Leu Arg Leu Ala Thr Glu Val - # Asn Trp Asp Glu Glu Lys Glu     Cys     #                670     -      Phe Glu Ser Leu Ser Lys Glu Cys - # Ala Met Phe Tyr Ser Ile Arg     Lys     #            685     -      Gln Tyr Ile Ser Glu Glu Ser Thr - # Leu Ser Gly Gln Gln Ser Glu     Val     #        700     -      Pro Gly Ser Ile Pro Asn Ser Trp - # Lys Trp Thr Val Glu His Ile     Val     #    720     -      Tyr Lys Ala Leu Arg Ser His Ile - # Leu Pro Pro Lys His Phe Thr     Glu     #   735     -      Asp Gly Asn Ile Leu Gln Leu Ala - # Asn Leu Pro Asp Leu Tyr Lys     Val     #                750     -      Phe Glu Arg Cys                  755     - (2) INFORMATION FOR SEQ ID NO:28:     -      (i) SEQUENCE CHARACTERISTICS:     #pairs    (A) LENGTH: 3065 base               (B) TYPE: nucleic acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: cDNA     -   (viii) POSITION IN GENOME:               (B) MAP POSITION: Chrom - #osome 5 Band G     #ID NO:28:(xi) SEQUENCE DESCRIPTION: SEQ     - CGGTGAAGGT CCTGAAGAAT TTCCAGATTC CTGAGTATCA TTGGAGGAGA CA - #GATAACCT       60     - GTCGTCAGGT AACGATGGTG TATATGCAAC AGAAATGGGT GTTCCTGGAG AC - #GCGTCTTT      120     - TCCCGAGAGC GGCACCGCAA CTCTCCCGCG GTGACTGTGA CTGGAGGAGT CC - #TGCATCCA      180     - TGGAGCAAAC CGAAGGCGTG AGTACAGAAT GTGCTAAGGC CATCAAGCCT AT - #TGATGGGA      240     - AGTCAGTCCA TCAAATTTGT TCTGGGCAGG TGATACTCAG TTTAAGCACC GC - #TGTGAAGG      300     - AGTTGATAGA AAATAGTGTA GATGCTGGTG CTACTACTAT TGATCTAAGG CT - #TAAAGACT      360     - ATGGGGTGGA CCTCATTGAA GTTTCAGACA ATGGATGTGG GGTAGAAGAA GA - #AAACTTTG      420     - AAGGTCTAGC TCTGAAACAT CACACATCTA AGATTCAAGA GTTTGCCGAC CT - #CACGCAGG      480     - TTGAAACTTT CGGCTTTCGG GGGGAAGCTC TGAGCTCTCT GTGTGCACTA AG - #TGATGTCA      540     - CTATATCTAC CTGCCACGGG TCTGCAAGCG TTGGGACTCG ACTGGTGTTT GA - #CCATAATG      600     - GGAAAATCAC CCAGAAAACT CCCTACCCCC GACCTAAAGG AACCACAGTC AG - #TGTGCAGC      660     - ACTTATTTTA TACACTACCC GTGCGTTACA AAGAGTTTCA GAGGAACATT AA - #AAAGGAGT      720     - ATTCCAAAAT GGTGCAGGTC TTACAGGCGT ACTGTATCAT CTCAGCAGGC GT - #CCGTGTAA      780     - GCTGCACTAA TCAGCTCGGA CAGGGGAAGC GGCACGCTGT GGTGTGCACA AG - #CGGCACGT      840     - CTGGCATGAA GGAAAATATC GGGTCTGTGT TTGGCCAGAA GCAGTTGCAA AG - #CCTCATTC      900     - CTTTTGTTCA GCTGCCCCCT AGTGACGCTG TGTGTGAAGA GTACGGCCTG AG - #CACTTCAG      960     - GACGCCACAA AACCTTTTCT ACGTTTTCGG GCTTCATTTC ACAGTGCACG CA - #CGGCGCCG     1020     - GGAGGAGTGC AACAGACAGG CAGTTTTTCT TCATCAATCA GAGGCCCTGT GA - #CCCAGCAA     1080     - AGGTCTCTAA GCTTGTCAAT GAGGTTTATC ACATGTATAA CCGGCATCAG TA - #CCCATTTG     1140     - TCGTCCTTAA CGTTTCCGTT GACTCAGAAT GTGTGGATAT TAATGTAACT CC - #AGATAAAA     1200     - GGCAAATTCT ACTACAAGAA GAGAAGCTAT TGCTGGCCGT TTTAAAGACC TC - #CTTGATAG     1260     - GAATGTTTGA CAGTGATGCA AACAAGCTTA ATGTCAACCA GCAGCCACTG CT - #AGATGTTG     1320     - AAGGTAACTT AGTAAAGTCG CATACTGCAG AACTAGAAAA GCCTGTGCCA GG - #AAAGCAAG     1380     - ATAACTCTCC TTCACTGAAG AGCACAGCAG ACGAGAAAAG GGTAGCATCC AT - #CTCCAGGC     1440     - TGAGAGAGGC CTTTTCTCTT CATCCTACTA AAGAGATCAA GTCTAGGGGT CC - #AGAGACTG     1500     - CTGAACTGAC ACGGAGTTTT CCAAGTGAGA AAAGGGGCGT GTTATCCTCT TA - #TCCTTCAG     1560     - ACGTCATCTC TTACAGAGGC CTCCGTGGCT CGCAGGACAA ATTGGTGAGT CC - #CACGGACA     1620     - GCCCTGGTGA CTGTATGGAC AGAGAGAAAA TAGAAAAAGA CTCAGGGCTC AG - #CAGCACCT     1680     - CAGCTGGCTC TGAGGAAGAG TTCAGCACCC CAGAAGTGGC CAGTAGCTTT AG - #CAGTGACT     1740     - ATAACGTGAG CTCCCTAGAA GACAGACCTT CTCAGGAAAC CATAAACTGT GG - #TGACCTGC     1800     - TGCCGTCCTC CAGGTACAGG ACAGTCCTTG AAGCCAGAAG ACCATGGATA TC - #AATGCAAA     1860     - GCTCTACCTC TAGCTCGTCT GTCACCCACA AATGCCAAGC GCTTCAAGAC AG - #AGGAAGAC     1920     - CCTCAAATGT CAACATATCT CAAAGATTGC CTGGTCCTCA GAGCACCTCA GC - #AGCTGAGG     1980     - TCGATGTAGC CATAAAAATG AATAAGAGAT CGTGCTCCTC GAGTTCTCTA GC - #TAAGCGAA     2040     - TGAAGCAGTT ACAGCACCTA AAGGCGCAGA ACAAACATGA ACTGAGTTAC AG - #AAAATTTA     2100     - GGGCCAAGAT TTGCCCTGGA GAAAACCAAG CAGCAGAAGA TGAACTCAGA AA - #AGAGATTA     2160     - GTAAATCGAT GTTTGCAGAG ATGGAGATCT TGGGTCAGTT TAACCTGGGA TT - #TATAGTAA     2220     - CCAAACTGAA AGAGGACCTC TTCCTGGTGG ACCAGCATGC TGCGGATGAG AA - #GTACAACT     2280     - TTGAGATGCT GCAGCAGCAC ACGGTGCTCC AGGCGCAGAG GCTCATCACG TG - #GGTGCACA     2340     - CAGGCTTCAG AGTTCCCAGA CCCCAGACTC TGAACTTAAC TGCTGTCAAT GA - #AGCTGTAC     2400     - TGATAGAAAA TCTGGAAATA TTCAGAAAGA ATGGCTTTGA CTTTGTCATT GA - #TGAGGATG     2460     - CTCCAGTCAC TGAAAGGGCT AAATTGATTT CCTTACCAAC TAGTAAAAAC TG - #GACCTTTG     2520     - GACCCCAAGA TATAGATGAA CTGATCTTTA TGTTAAGTGA CAGCCCTGGG GT - #CATGTGCC     2580     - GGCCCTCACG AGTCAGACAG ATGTTTGCTT CCAGAGCCTG TCGGAAGTCA GT - #GATGATTG     2640     - GAACGGCGCT CAATGCGAGC GAGATGAAGA AGCTCATCAC CCACATGGGT GA - #GATGGACC     2700     - ACCCCTGGAA CTGCCCCCAC GGCAGGCCAA CCATGAGGCA CGTTGCCAAT CT - #GGATGTCA     2760     - TCTCTCAGAA CTGACACACC CCTTGTAGCA TAGAGTTTAT TACAGATTGT TC - #GGTTCGCA     2820     - AAGAGAAGGT TTTAAGTAAT CTGATTATCG TTGTACAAAA ATTAGCATGC TG - #CTTTAATG     2880     - TACTGGATCC ATTTAAAAGC AGTGTTAAGG CAGGCATGAT GGAGTGTTCC TC - #TAGCTCAG     2940     - CTACTTGGGT GATCCGGTGG GAGCTCATGT GAGCCCAGGA CTTTGAGACC AC - #TCCGAGCC     3000     - ACATTCATGA GACTCAATTC AAGGACAAAA AAAAAAAGAT ATTTTTGAAG CC - #TTTTAAAA     3060     #          3065     - (2) INFORMATION FOR SEQ ID NO:29:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 864 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:29:(xi) SEQUENCE DESCRIPTION: SEQ     -      Met Glu Gln Thr Glu Gly Val Ser - # Thr Glu Cys Ala Lys Ala Ile     Lys     #   15     -      Pro Ile Asp Gly Lys Ser Val His - # Gln Ile Cys Ser Gly Gln Val     Ile     #                 30     -      Leu Ser Leu Ser Thr Ala Val Lys - # Glu Leu Ile Glu Asn Ser Val     Asp     #             45     -      Ala Gly Ala Thr Thr Ile Asp Leu - # Arg Leu Lys Asp Tyr Gly Val     Asp     #         60     -      Leu Ile Glu Val Ser Asp Asn Gly - # Cys Gly Val Glu Glu Glu Asn     Phe     #     80     -      Glu Gly Leu Ala Leu Lys His His - # Thr Ser Lys Ile Gln Glu Phe     Ala     #   95     -      Asp Leu Thr Gln Val Glu Thr Phe - # Gly Phe Arg Gly Glu Ala Leu     Ser     #                110     -      Ser Leu Cys Ala Leu Ser Asp Val - # Thr Ile Ser Thr Cys His Gly     Ser     #            125     -      Ala Ser Val Gly Thr Arg Leu Val - # Phe Asp His Asn Gly Lys Ile     Thr     #        140     -      Gln Lys Thr Pro Tyr Pro Arg Pro - # Lys Gly Thr Thr Val Ser Val     Gln     #    160     -      His Leu Phe Tyr Thr Leu Pro Val - # Arg Tyr Lys Glu Phe Gln Arg     Asn     #   175     -      Ile Lys Lys Glu Tyr Ser Lys Met - # Val Gln Val Leu Gln Ala Tyr     Cys     #                190     -      Ile Ile Ser Ala Gly Val Arg Val - # Ser Cys Thr Asn Gln Leu Gly     Gln     #            205     -      Gly Lys Arg His Ala Val Val Cys - # Thr Ser Gly Thr Ser Gly Met     Lys     #        220     -      Glu Asn Ile Gly Ser Val Phe Gly - # Gln Lys Gln Leu Gln Ser Leu     Ile     #    240     -      Pro Phe Val Gln Leu Pro Pro Ser - # Asp Ala Val Cys Glu Glu Tyr     Gly     #   255     -      Leu Ser Thr Ser Gly Arg His Lys - # Thr Phe Ser Thr Phe Ser Gly     Phe     #                270     -      Ile Ser Gln Cys Thr His Gly Ala - # Gly Arg Ser Ala Thr Asp Arg     Gln     #            285     -      Phe Phe Phe Ile Asn Gln Arg Pro - # Cys Asp Pro Ala Lys Val Ser     Lys     #        300     -      Leu Val Asn Glu Val Tyr His Met - # Tyr Asn Arg His Gln Tyr Pro     Phe     #    320     -      Val Val Leu Asn Val Ser Val Asp - # Ser Glu Cys Val Asp Ile Asn     Val     #   335     -      Thr Pro Asp Lys Arg Gln Ile Leu - # Leu Gln Glu Glu Lys Leu Leu     Leu     #                350     -      Ala Val Leu Lys Thr Ser Leu Ile - # Gly Met Phe Asp Ser Asp Ala     Asn     #            365     -      Lys Leu Asn Val Asn Gln Gln Pro - # Leu Leu Asp Val Glu Gly Asn     Leu     #        380     -      Val Lys Ser His Thr Ala Glu Leu - # Glu Lys Pro Val Pro Gly Lys     Gln     #    400     -      Asp Asn Ser Pro Ser Leu Lys Ser - # Thr Ala Asp Glu Lys Arg Val     Ala     #   415     -      Ser Ile Ser Arg Leu Arg Glu Ala - # Phe Ser Leu His Pro Thr Lys     Glu     #                430     -      Ile Lys Ser Arg Gly Pro Glu Thr - # Ala Glu Leu Thr Arg Ser Phe     Pro     #            445     -      Ser Glu Lys Arg Gly Val Leu Ser - # Ser Tyr Pro Ser Asp Val Ile     Ser     #        460     -      Tyr Arg Gly Leu Arg Gly Ser Gln - # Asp Lys Leu Val Ser Pro Thr     Asp     #    480     -      Ser Pro Gly Asp Cys Met Asp Arg - # Glu Lys Ile Glu Lys Asp Ser     Gly     #   495     -      Leu Ser Ser Thr Ser Ala Gly Ser - # Glu Glu Glu Phe Ser Thr Pro     Glu     #                510     -      Val Ala Ser Ser Phe Ser Ser Asp - # Tyr Asn Val Ser Ser Leu Glu     Asp     #            525     -      Arg Pro Ser Gln Glu Thr Ile Asn - # Cys Gly Asp Leu Leu Pro Ser     Ser     #        540     -      Arg Tyr Arg Thr Val Leu Glu Ala - # Arg Arg Pro Trp Ile Ser Met     Gln     #    560     -      Ser Ser Thr Ser Ser Ser Ser Val - # Thr His Lys Cys Gln Ala Leu     Gln     #   575     -      Asp Arg Gly Arg Pro Ser Asn Val - # Asn Ile Ser Gln Arg Leu Pro     Gly     #                590     -      Pro Gln Ser Thr Ser Ala Ala Glu - # Val Asp Val Ala Ile Lys Met     Asn     #            605     -      Lys Arg Ser Cys Ser Ser Ser Ser - # Leu Ala Lys Arg Met Lys Gln     Leu     #        620     -      Gln His Leu Lys Ala Gln Asn Lys - # His Glu Leu Ser Tyr Arg Lys     Phe     #    640     -      Arg Ala Lys Ile Cys Pro Gly Glu - # Asn Gln Ala Ala Glu Asp Glu     Leu     #   655     -      Arg Lys Glu Ile Ser Lys Ser Met - # Phe Ala Glu Met Glu Ile Leu     Gly     #                670     -      Gln Phe Asn Leu Gly Phe Ile Val - # Thr Lys Leu Lys Glu Asp Leu     Phe     #            685     -      Leu Val Asp Gln His Ala Ala Asp - # Glu Lys Tyr Asn Phe Glu Met     Leu     #        700     -      Gln Gln His Thr Val Leu Gln Ala - # Gln Arg Leu Ile Thr Trp Val     His     #    720     -      Thr Gly Phe Arg Val Pro Arg Pro - # Gln Thr Leu Asn Leu Thr Ala     Val     #   735     -      Asn Glu Ala Val Leu Ile Glu Asn - # Leu Glu Ile Phe Arg Lys Asn     Gly     #                750     -      Phe Asp Phe Val Ile Asp Glu Asp - # Ala Pro Val Thr Glu Arg Ala     Lys     #            765     -      Leu Ile Ser Leu Pro Thr Ser Lys - # Asn Trp Thr Phe Gly Pro Gln     Asp     #        780     -      Ile Asp Glu Leu Ile Phe Met Leu - # Ser Asp Ser Pro Gly Val Met     Cys     #    800     -      Arg Pro Ser Arg Val Arg Gln Met - # Phe Ala Ser Arg Ala Cys Arg     Lys     #   815     -      Ser Val Met Ile Gly Thr Ala Leu - # Asn Ala Ser Glu Met Lys Lys     Leu     #                830     -      Ile Thr His Met Gly Glu Met Asp - # His Pro Trp Asn Cys Pro His     Gly     #            845     -      Arg Pro Thr Met Arg His Val Ala - # Asn Leu Asp Val Ile Ser Gln     Asn     #        860     - (2) INFORMATION FOR SEQ ID NO:30:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 862 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: protein     #ID NO:30:(xi) SEQUENCE DESCRIPTION: SEQ     -      Met Glu Arg Ala Glu Ser Ser Ser - # Thr Glu Pro Ala Lys Ala Ile     Lys     #   15     -      Pro Ile Asp Arg Lys Ser Val His - # Gln Ile Cys Ser Gly Gln Val     Val     #                 30     -      Leu Ser Leu Ser Thr Ala Val Lys - # Glu Leu Val Glu Asn Ser Leu     Asp     #             45     -      Ala Gly Ala Thr Asn Ile Asp Leu - # Lys Leu Lys Asp Tyr Gly Val     Asp     #         60     -      Leu Ile Glu Val Ser Asp Asn Gly - # Cys Gly Val Glu Glu Glu Asn     Phe     #     80     -      Glu Gly Leu Thr Leu Lys His His - # Thr Ser Lys Ile Gln Glu Phe     Ala     #   95     -      Asp Leu Thr Gln Val Glu Thr Phe - # Gly Phe Arg Gly Glu Ala Leu     Ser     #                110     -      Ser Leu Cys Ala Leu Ser Asp Val - # Thr Ile Ser Thr Cys His Ala     Ser     #            125     -      Ala Lys Val Gly Thr Arg Leu Met - # Phe Asp His Asn Gly Lys Ile     Ile     #        140     -      Gln Lys Thr Pro Tyr Pro Arg Pro - # Arg Gly Thr Thr Val Ser Val     Gln     #    160     -      Gln Leu Phe Ser Thr Leu Pro Val - # Arg His Lys Glu Phe Gln Arg     Asn     #   175     -      Ile Lys Lys Glu Tyr Ala Lys Met - # Val Gln Val Leu His Ala Tyr     Cys     #                190     -      Ile Ile Ser Ala Gly Ile Arg Val - # Ser Cys Thr Asn Gln Leu Gly     Gln     #            205     -      Gly Lys Arg Gln Pro Val Val Cys - # Thr Gly Gly Ser Pro Ser Ile     Lys     #        220     -      Glu Asn Ile Gly Ser Val Phe Gly - # Gln Lys Gln Leu Gln Ser Leu     Ile     #    240     -      Pro Phe Val Gln Leu Pro Pro Ser - # Asp Ser Val Cys Glu Glu Tyr     Gly     #   255     -      Leu Ser Cys Ser Asp Ala Leu His - # Asn Leu Phe Tyr Ile Ser Gly     Phe     #                270     -      Ile Ser Gln Cys Thr His Gly Val - # Gly Arg Ser Ser Thr Asp Arg     Gln     #            285     -      Phe Phe Phe Ile Asn Arg Arg Pro - # Cys Asp Pro Ala Lys Val Cys     Arg     #        300     -      Leu Val Asn Glu Val Tyr His Met - # Tyr Asn Arg His Gln Tyr Pro     Phe     #    320     -      Val Val Leu Asn Ile Ser Val Asp - # Ser Glu Cys Val Asp Ile Asn     Val     #   335     -      Thr Pro Asp Lys Arg Gln Ile Leu - # Leu Gln Glu Glu Lys Leu Leu     Leu     #                350     -      Ala Val Leu Lys Thr Ser Leu Ile - # Gly Met Phe Asp Ser Asp Val     Asn     #            365     -      Lys Leu Asn Val Ser Gln Gln Pro - # Leu Leu Asp Val Glu Gly Asn     Leu     #        380     -      Ile Lys Met His Ala Ala Asp Leu - # Glu Lys Pro Met Val Glu Lys     Gln     #    400     -      Asp Gln Ser Pro Ser Leu Arg Thr - # Gly Glu Glu Lys Lys Asp Val     Ser     #   415     -      Ile Ser Arg Leu Arg Glu Ala Phe - # Ser Leu Arg His Thr Thr Glu     Asn     #                430     -      Lys Pro His Ser Pro Lys Thr Pro - # Glu Pro Arg Arg Ser Pro Leu     Gly     #            445     -      Gln Lys Arg Gly Met Leu Ser Ser - # Ser Thr Ser Gly Ala Ile Ser     Asp     #        460     -      Lys Gly Val Leu Arg Ser Gln Lys - # Glu Ala Val Ser Ser Ser His     Gly     #    480     -      Pro Ser Asp Pro Thr Asp Arg Ala - # Glu Val Glu Lys Asp Ser Gly     His     #   495     -      Gly Ser Thr Ser Val Asp Ser Glu - # Gly Phe Ser Ile Pro Asp Thr     Gly     #                510     -      Ser His Cys Ser Ser Glu Tyr Ala - # Ala Ser Ser Pro Gly Asp Arg     Gly     #            525     -      Ser Gln Glu His Val Asp Ser Gln - # Glu Lys Ala Pro Glu Thr Asp     Asp     #        540     -      Ser Phe Ser Asp Val Asp Cys His - # Ser Asn Gln Glu Asp Thr Gly     Cys     #    560     -      Lys Phe Arg Val Leu Pro Gln Pro - # Thr Asn Leu Ala Thr Pro Asn     Thr     #   575     -      Lys Arg Phe Lys Lys Glu Glu Ile - # Leu Ser Ser Ser Asp Ile Cys     Gln     #                590     -      Lys Leu Val Asn Thr Gln Asp Met - # Ser Ala Ser Gln Val Asp Val     Ala     #            605     -      Val Lys Ile Asn Lys Lys Val Val - # Pro Leu Asp Phe Ser Met Ser     Ser     #        620     -      Leu Ala Lys Arg Ile Lys Gln Leu - # His His Glu Ala Gln Gln Ser     Glu     #    640     -      Gly Glu Gln Asn Tyr Arg Lys Phe - # Arg Ala Lys Ile Cys Pro Gly     Glu     #   655     -      Asn Gln Ala Ala Glu Asp Glu Leu - # Arg Lys Glu Ile Ser Lys Thr     Met     #                670     -      Phe Ala Glu Met Glu Ile Ile Gly - # Gln Phe Asn Leu Gly Phe Ile     Ile     #            685     -      Thr Lys Leu Asn Glu Asp Ile Phe - # Ile Val Asp Gln His Ala Thr     Asp     #        700     -      Glu Lys Tyr Asn Phe Glu Met Leu - # Gln Gln His Thr Val Leu Gln     Gly     #    720     -      Gln Arg Leu Ile Ala Pro Gln Thr - # Leu Asn Leu Thr Ala Val Asn     Glu     #   735     -      Ala Val Leu Ile Glu Asn Leu Glu - # Ile Phe Arg Lys Asn Gly Phe     Asp     #                750     -      Phe Val Ile Asp Glu Asn Ala Pro - # Val Thr Glu Arg Ala Lys Leu     Ile     #            765     -      Ser Leu Pro Thr Ser Lys Asn Trp - # Thr Phe Gly Pro Gln Asp Val     Asp     #        780     -      Glu Leu Ile Phe Met Leu Ser Asp - # Ser Pro Gly Val Met Cys Arg     Pro     #    800     -      Ser Arg Val Lys Gln Met Phe Ala - # Ser Arg Ala Cys Arg Lys Ser     Val     #   815     -      Met Ile Gly Thr Ala Leu Asn Thr - # Ser Glu Met Lys Lys Leu Ile     Thr     #                830     -      His Met Gly Glu Met Gly His Pro - # Trp Asn Cys Pro His Gly Arg     Pro     #            845     -      Thr Met Arg His Ile Ala Asn Leu - # Gly Val Ile Ser Gln Asn     #        860     __________________________________________________________________________ 

We claim:
 1. A purified mouse mPMS1 gene shown in SEQ ID NO:28.
 2. Isolated and purified DNA comprising a segment of at least 13 nucleotides of mPMS1 as shown in SEQ ID NO:28.
 3. An isolated polynucleotide comprising at least two separate segments, each segment having a sequence of at least 13 nucleotides the same as any 13 nucleotide sequence in SEQ ID NO: 28, wherein the sequences of the segments can be used to design a pair of olizonucleotide primers for amplifying specifically at least a portion of mPMS1. 